Anti-tumor molecular vaccine and method of making thereof

ABSTRACT

The present invention provides a vaccine which is capable of inhibiting the growth and formation of tumors which can express an endogenous tumor specific protein. Also disclosed are methods of administering the vaccine to a subject bearing a tumor whose formation and growth is inhibited by the vaccine.

FIELD OF THE INVENTION

[0001] The present invention relates to a novel anti-tumor vaccine,including anti-EFGR molecular vaccine. The field of molecular vaccineencompasses many aspects of biotechnology, such as PCR, MolecularCloning, Gene Expression, Modern vaccine technology, andbiotechnological medicine and pharmacology.

BACKGROUND

[0002] Vaccines are materials that are capable of stimulating the immunesystem to initiate an immune response against specific target substancessuch as virus or bacteria. Classical concept of vaccine originated fromthe process of generating immunity against infectious diseases. Thatprocess mainly consisted of using treated microbial pathogen (such asvirus and bacteria) or its derivatives to immunize the organism so as toproduce humoral immune response to prevent the onset of infectiousdiseases. For example, inactivated vaccine is derived by inactivatingthe active component of the infectious pathogen. Attenuated vaccine isproduced by mutating live virus or bacteria so that it cannot reproduceitself in biological organism. These two classes of vaccines achieve thegoal of immunization through the surface antigen of the pathogen andtheir reaction with B cells and T cells.

[0003] Cancer vaccines are of particular importance in cancer therapy.This type of vaccine is different from traditional, preventive vaccine,in that, it is mainly used on patients who are already inflicted withcancer. The purpose of using cancer vaccine is to stimulate thepatient's specific immune response against the cancer, to the point thateventually the cancer is effectively rejected by the organism, andtherefore cured. The research and development of cancer vaccine hasbecome a focal point of cancer therapy internationally. Cancer vaccinesmainly include cancer cell vaccine, gene-modified vaccine, polypeptidetumor vaccine, and gene/DNA vaccine, etc.

[0004] Cancer cell vaccines are intact, dead cells produced by treatingcancer cells of a patient or animal with physical or chemical methods,such that after treatment, those cells will possess therapeutic orauxiliary therapeutic effect. The methods used to treat the cancer cellsinclude X-ray radiation, or treating with organic solvent, etc. Afterintroducing this vaccine to the patient using injection, or othermethod, the cancer cell vaccine can stimulate or enhance the patient'simmune response against the targeted cancer. Genetically-modifiedvaccine, polypeptide cancer vaccine, and gene/DNA vaccines are allvaccines having therapeutic effect on the targeted cancer, and made byusing the cancer antigen or its fragments, or polynucleotides coding forsuch cancer antigen or its fragments, and carriers/cells containing thepolynucleotides.

[0005] Cancer vaccine studies have become an important area in the fightto cure cancer and save the lives of thousands of patients worldwide. Ithas been recognized in the medical research and clinical studies thatone of the key factor for the success of any cancer therapy is itsability to distinguish neoplastic cells, which should be killed by thetherapy, from normal cells, which should be unaffected, and left aloneas much as possible, by the therapy. In reality, though, it is alsodifficult, if not impossible, to achieve this goal. Many availablecancer therapy protocols are based upon the differential growth rate ofcancer cells, that is to say, cancer cells tend to divide andproliferate much faster than normal cells. But, such cancer therapieswould not be effective in the case of cancers such as brain tumor. Anideal situation is to use a tumor specific antigen which is exclusivelyexpressed on tumor cells to immunize the patient or subject animal,because the immune system would most efficiently recognize an antigenthat is has never seen before. In that case, it would be possible to useone's own immune system to destroy a cancer. Under this reasoning, thesuccess rate of the approach would depend upon the identification of anappropriate antigen that can elicit a humoral or cellular immuneresponse against the targeted tumor.

[0006] Research developed over the years in the past has uncovered manytumor specific proteins and molecules that are specifically related tothe onset of a particular cancer. These molecules are generally referredherein as tumor-specific proteins. Many of the TSPs are good candidatesfor tumor antigen. One of such TSP is epidermal growth factor receptor,EGFR.

[0007] EGFR is a trans-membrane, single chain, glycosylated protein witha molecular weight of 170 KD. It is consisted of 1186 amino acidresidues, and possesses Tyrosine Protein Kinase (PTK) activity. Thestructure of the EGFR molecule is consisted of three main parts: anextracellular domains a transmembrane domain, and an intracellulardomain. The ligand binding activity occurs in the extracellular domainand the PTK activity resides within the intracellular domain. Itsligand, EGF, or TGF-α can act on EFGR through either autocrine pathway,or paracrine pathway, by activating PTK, and through a series of signaltransduction, causing cells to divide and proliferate. EFGR can bewidely found on the surface of normal mammalian epithelial cell surface.On average, there are about 50-100 thousand receptor molecules per cell.Many cancer cells such as lung cancer, breast cancer, ovary cancer,colon cancer, prostate cancer, bladder cancer, head and necksquamocarcinoma and glioma, all have over-expressed EGFR, up to 1-3×10⁶molecules. Take, as an example, in the case of lung cancer, theoverexpression of EGFR is closely related to the cancer's infiltration,metastasis and prognosis. Hence, EGFR has been commonly accepted as atumor specific antigen. Logically, EGFR has been hailed as one of theideal target molecules in cancer therapy. So far, however, the majorapplication of using EGFR as a therapy target has been with monoclonalantibody and small molecular synthetic compounds. There has been somepositive development in areas such as using EGFRvIII molecule in tumorpeptide vaccine, and anti-sense RNA gene therapy. Nonetheless, therehave been few reports in literature regarding using EGFR as a targetmolecule in making anti-tumor molecular vaccine. Thus, there exists aneed to develop new vaccines to specifically target tumor antigens suchas EGFR in order to inhibit the growth and formation of tumors.

SUMMARY OF THE INVENTION

[0008] The present invention provides anti-tumor tumor specific protein(TSP) molecular vaccine, including autologous TSP varian vaccine,xenogeneic TSP vaccine, and gene directed evolution TSP vaccine. As apreferred embodiment, such TSP is a tumor receptor protein. A furtherpreferred embodiement of the TSP molecular vaccine is the EpidermalGrowth Factor Receptor (EFGR) molecular vaccine. TSP molecular vaccinesencompass a variety of biological vaccine and they can be made using asantigen a xenogeneic homologous TSP molecule that is either geneticallyengineered, mutated, and improved, or, derived from human or otherdifferent species due to natural evolution. TSP molecular vaccineincludes recombinant protein vaccine, recombinant gene vaccine,recombinant viral vaccine, modified gene vaccine, and stabletransformants of commensal bacteria. Tumor receptor vaccine is a newtype of tumor vaccine.

[0009] An object of the present invention is to provide a vaccine whichcomprises a molecular homolog having sufficient structural similarity toa tumor specific protein endogenously expressed in a tumor such that themolecular homolog is capable of inducing an immune response to the tumorspecific protein in a subject bearing the tumor.

[0010] Another object of the invention is to provide a method forinducing immunity against a tumor specific protein endogenouslyexpressed in a tumor which comprises administering to a subject bearingthe tumor a molecular homolog having sufficient structural similarity tothe tumor specific protein so as to enable the molecular homolog toinduce the immunity against the tumor specific protein.

[0011] A further object of the invention is to provide a method ofimmunizing an animal, preferably a human, against a tumor having a tumorspecific protein endogenously expressed therein, comprising the step ofadministering to the animal a molecular homolog having sufficientstructural similarity to the tumor specific protein so as to enable themolecular homolog to induce an immune response to the tumor specificprotein.

[0012] Yet another object of the invention is to provide a method ofmaking a vaccine for inducing an immune response against a tumorspecific protein endogenously expressed in a tumor, which comprisesselecting a molecular homolog having sufficient structural similarity tothe tumor specific protein so as to enable the molecular homolog toinduce the immune response against the tumor specific protein in thesubject bearing the tumor.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 is a schematic diagram of the mechanism of action ofhomologous molecular vaccine.

[0014]FIG. 2 shows recombinant EGFR plasmid maps. 2A shows recombinantEGFR eukaryotic expression plasmid maps; 2B shows recombinant EGFRprokaryotic expression plasmid maps; 2C shows recombinant EGFR yeastexpression plasmid maps; 2D shows recombinant EGFR Adenovirus shuttleplasmid maps; 2E shows recombinant EGFR Lentivirus precursor plasmidmaps.

[0015]FIG. 3 is a flow chart depicting the construction of EGFRrecombinant protein vaccine.

[0016]FIG. 4 is a flow chart depicting the construction of EGFRrecombinant virus vaccine. FIG. 4A is a flow chart depicting theconstruction of EGFR recombinant Adenovirus vaccine; FIG. 4B is a flowchart depicting the construction of EGFR recombinant Lentivirus vaccine.

[0017]FIG. 5 is a flow chart depicting the construction of theRGD-modified EGFR recombinant Adenovirus vaccine.

[0018]FIG. 6, including FIGS. 6A, 6B and 6C, is a schematic diagramdepicting the mechanism of action for the nanoparticle targeted EGFRmolecular vaccine.

[0019]FIG. 7 is a graph showing the induction of protective anti-tumorimmunity of EGFR recombinant DNA vaccine. 7A. Tumor Volume changes ofimmunized mice bearing LL/2c Lewis lung cancer. 7B. Tumor Volume changesof immunized mice bearing MA782/5S mammary cancer. 7C. Survival rate ofimmunized mice bearing LL/2c Lewis lung cancer. 7D. Survival rate ofimmunized mice bearing MA782/5S mammary cancer. hEe-p, human EGFRextracellular DNA vaccine; mEe-p, mouse EGFR extracelluar DNA vaccine;c-p, blank plasmid control; Saline, saline control.

[0020]FIG. 8 is a graph showing the induction of therapeutic anti-tumorimmunity of EGFR recombinant DNA vaccine. 8A. Tumor Volume changes ofimmunized mice bearing LL/2c Lewis lung cancer. 8B. Tumor Volume changesof immunized mice bearing MA782/5S mammary cancer. 8C. Survival rate ofimmunized mice bearing LL/2c Lewis lung cancer. 8D. Survival rate ofimmunized mice bearing MA782/5S mammary cancer. hEe-p, human EGFRextracellular DNA vaccine; mEe-p, mouse EGFR extracelluar DNA vaccine;c-p, blank plasmid control; Saline, saline control.

[0021]FIG. 9 depicts the induction of anti-tumor immunity of EGFRrecombinant protein vaccine. 9A. Protective immunity. 9B. Therapeuticimmunity. 9C. Survival curves for mice bearing MA782/5S mammary cancer.edCER: Recombinant protein vaccine containing Chicken EGFR extracellulardomain. edMER: Recombinant protein vaccine containing Mouse EGFRextracellular domain. Adj: Adjuvant. NS: Saline.

[0022]FIG. 10 depicts the anti-tumor metastasis effect of EGFRrecombinant protein vaccine. 10A. Number of transferer of LL/2c lungcancer. 10B. Lung wet weight after LL/2c tumor metastasis. edCER:Recombinant protein vaccine containing Chicken EGFR extracellulardomain. edMER: Recombinant protein vaccine containing Mouse EGFRextracellular domain. Adj: Adjuvant. NS: Saline.

[0023]FIG. 11 depicts EGFR molecular vaccine's inhibition of tumor cellgrowth in vitro, and its adoptive anti-tumor immunity. 11A. Theinhibition of growth of EGFR-positive tumor cells (A549, LL/2c,MA782/5S) and EGFR-negative tumor cells (H22 and MMT-06052) by Igderived from mice immunized with EGFR Recombinant DNA vaccine hEe-p.11B. Inhibition of growth of tumor cells by Ig derived fromnon-immunized normal mice. 11C. In vivo adoptive anti-tumor immunity ofIg derived from mice immunized with EGFR recombinant DNA vaccine hEe-p.hEe-p, human EGFR extracellular DNA vaccine; mEe-p, mouse EGFRextracelluar DNA vaccine; c-p, blank plasmid control; Saline, salinecontrol.

[0024]FIG. 12 depicts the types of antibodies induced by EGFR molecularvaccine. 12A. Types of antibodies induced by EGFR recombinant DNAvaccine. 12B. Types of antibodies induced by EGFR recombinant protein.vaccine. hEe-p, human EGFR extracellular DNA vaccine; mEe-p, mouse EGFRextracelluar DNA vaccine; c-p, blank plasmid control; Saline, salinecontrol. edCER: Recombinant protein vaccine containing Chicken EGFRextracellular domain. edMER: Recombinant protein vaccine containingMouse EGFR extracellular domain. Adj: Adjuvant. NS: Saline.

[0025]FIG. 13 depecits the induction of CTL-mediated cytotoxicity invitro with EGFR DNA vaccine and adoptive transfer of T cell subsets. A,T cells derived from the spleens of hEe-p-immunized mice were testedagainst LL/2c cells at different E:T ratios by a standard ⁵¹Cr releaseassay. T cells derived from the spleens of hEe-p-immunized mice showedhigher cytotoxicity against LL/2c cells than did T cells from mEe-p,c-p, or nonimmunized mice by a standard ⁵¹Cr release assay.hEe-p-induced tumor killing activity can be blocked by anti-CD8 oranti-MHC class I (anti-H-2 Kb/H-2 Db) mAb. B and C, T cells wereisolated from spleens of C57BL/6 mice, immunized with hEe-p ( ), mEe-p,and nonimmunized mice ( ), and were depleted of CD4+ or CD8+lymphocytes. The adoptive transfer of 2×10⁷ CD4-depleted (CD8+) (B) orCD8-depleted (CD4+) (C) T cells from mice immunized with hEe-p showedthe antitumor activity against EGFrpositive LL/2c. D and E, The adoptivetransfer of 2×10⁷ CD4-depleted (CD8+)(D) or CD8-depleted (CD4+) (E) Tcells from BALB/c mice immunized with hEe-p was also found to beeffective in EGFr-positive MA782/5S mammary cancer model. There was noantitumor activity found in syngeneic EGFr-negative tumor (B16 and MethA) (B-E). In addition, the transfer of T lymphocyte subsets from miceimmunized with mEe-p and nonimmunized mice ( ) had no effect (B-E). Datarepresent day 25 after tumor cell injection.

[0026]FIG. 14 depicts the identification of auto-antibodies on the tumorcells by fluorescence microscopy. There was the deposition ofauto-antibodies on LL/2c tumor cells (A) and MA782/5S tumor cells (B)from hEe-p-immunized mice, but not on the corresponding tumor cells fromnon-immunized mice (C and D). The mice depleted of CD4+ T lymphocyteswere immunized with hEe-p and did not develop detectable IgG-specificfluorescence on LL/2c tumor cells (E) and MA782/5S tumor cells the tumorcells (F). In contrast, the depletion of CD8+ lymphocytes (G and H) orNK cells showed no effect. There was no deposition of auto-antibodieswithin the tissues of the liver (I) and kidney (J) of mice immunizedwith hEe-p and the corresponding tissues (K and L) from non-immunizedmice.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention deals with a new type of anti-cancervaccine—tumor specific protein molecular vaccine, such as tumor receptorvaccine, i.e. EGFR molecular vaccine. The description hereinafter isprovided in some detail with respect to an embodiment of the invention,namely, EGFR molecular vaccine, and its use in animal tumor model.However, as discussed above, the practices of the present invention canalso be applied to the production and use of other tumor-specificreceptor such as VEGF, TGF-β, etc.

[0028] In the description of the invention set forth hereinabove,emphasis has been placed upon the preparation of a vaccine based uponthe EGFR molecule. It is clearly indicated, however, that the conceptand practices of this invention are generally applicable to thepreparation of vaccines based upon other tumor associated proteinsincluding other tumor specific growth factor receptors to treat orprevent human cancers and for the preparation of vaccines to treat orprevent infectious diseases in man and animals.

[0029] In order to confirm the hypothesis mentioned above, we selectedsome cancer cell proliferation-associated molecules (such as EGFR,insulin-like growth factor receptor (IGFR), etc) or tumorangiogenesis-associated molecules (such as vascular endothelial growthfactor (VEGF), αvβ3 integrin, endoglin, vascular endothelial growthfactor receptor (KDR), fibroblastic growth factor receptor (FGFR) andTie2, etc., from Xenopus laevis, bird, mouse, pig, bovine, even from thefruit fly Drosophila melanogaster as target molecules. We isolated thecounterparts of these homologous genes, immunized mouse models with theplasmid DNA vector or adenoviral vector inserted with these xenogeneicgenes or their recombinant proteins or synthetic peptides, observedtheir activity of anti-tumors (including mammary cancer, lung cancer,melanoma, hepatocarcinoma and fibrosarcoma) and explored the possiblemolecular and immunological mechanisms.

[0030] As used herein, the term “tumor specific protein” refers to aprotein which is specifically associated with a particular tumor, and isnot usually found in normal cells, or if it is found in normal cells, itis uncontroallable overexpressed in the tumor, as if often the case. Onegroup of tumor specific protein (TSP) is tumor receptor protein which,after being bound by a ligand molecule, activates a cellular mechanismto enable the tumor cells to growth uncontrollably. Examples of a tumorreceptor protein include Epidermal Growth Factor Receptor, EGFR,insulin-like growth factor receptor (IGFR). Other examples of TSPinclude tumor angiogenesis-associated molecules such as vascularendothelial growth factor (VEGF), αvβ3 integrin, endoglin, vascularendothelial growth factor receptor (KDR), fibroblastic growth factorreceptor (FGFR) and Tie2, etc.

[0031] As used herein, the term “molecular homolog” means either a DNAmolecule, or, a protein or polypeptide molecule, which share seqeuncesimilarity with another corresponding molecule.

[0032] As used herein, the term “structural similarity” refers tosequences similarity between two DNA molecules, or two Proteinmolecules, respective. Such similarity can be deduced using industrystandard tools such as BLAST and other bioinformatical software.

[0033] As used herein, the term “endogenously expressed” refers to agene that is found to be in existence in the native cell, and notintroduced through a foreign vector.

[0034] As used herein, the term “a subject” refers to an animal,including a human, mouse, a bird, a chick, nematode, etc.

[0035] As used herein, the term “an immune response” refers to areaction by the subject to fight against a recognized antigen, thereacion can be the generation of antibody which specifically binds theantigen, or the reaction can be a cytotoxic T-Lymphocyte activity (CTL)

[0036] As used herein, the term “bearing” means that the subject carriesthe tumor within its organs, tissues, etc.

[0037] As used herein, the term “xenogeneic homolog” refers to a proteinor a gene which is derived from an animal of different species from themolecule of similar biological function that it's compared to.

[0038] As used herein, the term “genetic engineering” refers to varioustechniques that can be used to generate DNA or protein homolog, such aserror-prone PCR, random primer extension technique, and DNA shufflingtechnique, which can cause artificial mutation in autologous EGFRmolecule, and then establish a gene mutation library.

[0039] 1. Origin of Egfr Molecule, its Selection and Improvement

[0040] The term EGFR molecule as used herein includes modifiedautologous EGFR molecule, and xenogeneic EGFR molecule, andgene-directed evolution EGFR molecule. A key feature of the presentinvention is demonstrated in the use of xenogeneic homologous EGFRmolecule as a molecular antigen in anti-tumor immunotherapy, said EGFRmolecular vaccine is made either with genetic engineering, or derivedfrom the natural evolutionary process, and the differences between thesequence of the molecular antigen and sequence of the EGFR of the targetsubject, such as an animal or human is utilized to illicit an immuneresponse against the EGFR of the target subject.

[0041] EGFR is widely found in nature in a variety of organisms rangingfrom mammals such as human, mouse, to avian organism such birds andchickens, to lower organism such as nematode, and fruit fly. The EGFRmolecules from these different organisms have certain differencesamongst them. Their homology amongst each other is between 30% to 100%.The homology between the xenogeneic EGFR molecules as disclosed in thepresent invention is between 45% to 95%, and as examples, we used human,mice, chicken, and Drosophila as exemplary species to discuss theinvention. At the amino acid level, the homology between mouse EGFR andhuman, chicken, and Drosophila is 87%, 72%, and 55%.

[0042] Comparative genomics pointed out that there are 70 to 1100thousands of genes in all mammals, a certain number of which are ratherconserved. These genes show certain degree of similarity in structuresand/or functions between different species. They are xenogeneichomologous genes. For example, epidermal growth factor receptor (EGFR)genes of human have the homology of 88%˜93% with mice, 72%˜83% withbirds, and 40%˜56% with fruit flies.

[0043] One of the classic problems in using tumor receptor as antigen inimmunotherapy is that autologous EGFR molecule is immuno-tolerant by thehost body, and thus its immunogenicity is very weak, if at all. By usingmodern biotechnological engineering, autologous EGFR molecules can bemodified with the procedure of gene directed evolution, and thusimproved and enhance the immunogenicity of the EGFR antigen. Thetechnical protocol is generally using error-prone PCR, random primerextension technique, and DNA shuffling technique, and cause artificialmutation in autologous EGFR molecule, and then establish a gene mutationlibrary. After that, undertake a process of selection using phagedisplay technique, ribosome display technique, and obtain EGFR moleculeswith stronger immunogenicity. In addition, we can utilize thedifferences between the expression modification systems of bacteria,virus and other organisms, and further modify and improve the autologousEGFR molecules at the protein level, in order to increase theimmunogenicity of autologous EGFR.

[0044] It has been known that tumor cells would produce one or morekinds of tumor antigens during its course of malignant transformationand proliferation. However, in most cases, tumor antigen isdifferentiation antigen with very weak immunogenicity that is not enoughto induce active immune response. In addition, in the view ofimmunology, tumor cells themselves are the cells of host, which cancontinuously express “normal” antigen (gene overexpression) and/orabnormal antigen (resulting from gene modification, mutation ordeletion). We can therefore consider tumor antigen as self-antigen inthis sense. In normal physiological state, a body cannot evoke immuneresponse to self-antigen, which is immune tolerance. In fact,self-antigen is the most compatible and the richest antigen to which thehost immune system must tolerate. The induction and maintenance ofself-antigen is mediated by several kinds of mechanisms by which thenormal tissues can be protected from improper injury. However, when thehost cryptic antigen releases or changes by some kinds of biological,physical or chemical reasons, it can induce autologous immune response,which acts on the tissues or cells the target antigen located andresults in pathological changes and blocking the function of thecorresponding organelle. So, if we can release the cryptic self-antigenof tumor cells or modify it to somewhat, we could therefore inducespecific autologous immune response to autologous tumor cells, andconsequently tumor regression or suppression. In other words, we cantake cancer immunotherapy by inducing autologous immune response of abody.

[0045] The cDNA sequence corresponding to normal EGF receptor has beenreported by Ullrich et al., in Nature 1984 309, 418-425, Ullrich, A.,Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam, A. W., Lee,J., Yarden, Y., Libermann, T. A., Schlessinger, J., et al. (1984). HumanEpidermal Growth Factor Receptor cDNA Sequence and Aberrant Expressionof the Amplified Gene in A431 Epidermoid Carcinoma Cells. Nature309:418-425, and was characterized the genetic alterations associatedwith rearrangements or deletions of this gene in five malignant gliomas.EGF receptor gene is expressed on the cell surface (Humphrey et al.,Cancer Research 1988, 48, 2231-2238). The EGF receptor gene has beenshown to be amplified in 40% of glioblastoma multiform tumors (Libermannet al., Nature 1985, 313(5998), 144-7; Wong et al., Proc Natl Acad SciUSA 1987 84(19), 6899-903). This receptor has been implicated in a widevariety of tumors including those of the breast, skin and bladder(Harris, A. L. Recent Results in Cancer Research 1989, 113, 70-77).

[0046] It has been known that tumor cells would produce one or moretypes of tumor antigens during its course of malignant transformationand proliferation. However, in most cases, tumor antigen isdifferentiation antigen with very weak immunogenicity that is not enoughto induce active immune response. In addition, in the view ofimmunology, tumor cells themselves are the cells of host, which cancontinuously express “normal” antigen (gene overexpression) and/orabnormal antigen (resulting from gene modification, mutation ordeletion). We can therefore consider tumor antigen as self-antigen inthis sense. In normal physiological state, a body cannot evoke immuneresponse to self-antigen, which is immune tolerance. In fact,self-antigen is the most compatible and the richest antigen to which thehost immune system must tolerate. The induction and maintenance ofself-antigen is mediated by several kinds of mechanisms by which thenormal tissues can be protected from improper injury. However, when thehost cryptic antigen releases or changes by some kinds of biological,physical or chemical reasons, it can induce autologous immune response,which acts on the tissues or cells the target antigen located andresults in pathological changes and blocking the function of thecorresponding organelle. So, if we can release the cryptic self-antigenof tumor cells or modify it to somewhat, we could therefore inducespecific autologous immune response to autologous tumor cells, andconsequently tumor regression or suppression. In other words, we cantake cancer immunotherapy by inducing autologous immune response of abody.

[0047] In general, cancer vaccines are based on the weak immunogenicityof target tumor antigen mixed with adjuvant in order to produce, recoveror enhance anti-cancer immune response and kill the residual or invasivetumor cells. The potential target of anti-self-antigen or anti-tumorincludes over expressed protein, tissue-specific differentiationantigen, development protein which tumor cells abnormally expressed, andso on. How to enhance the immunogenicity of target antigen then?

[0048] As we mentioned above, there exist a certain number of xenogeneichomologous genes between various species. It is commonly expected thatthe xenogeneic homologous genes between various species should have someimmunological significance in the course of evolution. One could takeadvantage of the subtle differences of xenogeneic homologous genesderived from evolution to break immune tolerance, enhance immunogenicityand induce autologous immune response of tumor cells, which leading tothe killing of tumor cells. The mechanism of which probably as follows:although the neutral mutation of xenogeneic homologous gene fromevolution does not lose or change its function, it probable affects orchanges its mode of immune response. When xenogeneic homologous genesare introduced into a host and expressed corresponding xenogeneichomologous protein, the host will recognize it as foreign antigen andeliminate it by producing the specific antibodies or CTL, on the otherhand, it will lead non-specific cross immune reaction because of thesimilarity between xenogeneic homologous proteins and the relatedprotein in the host, and thereby inducing autologous immune response andbreaking the immune tolerance of the body to this protein. Maybe theimmunological rejection during heterogenous transplantation is also dueto the existence of xenogeneic homologs (such as genes, peptides orproteins).

[0049] The gene-directed evolution technology refers to the method ofusing artificial technique to change the characteristics of a particulargene in a significant way, for example, to change an enzyme with lowcatalytic activity in a reaction to enable the enzyme to become highlycatalytic in the reaction. The process of gene-directed evolution speedsup the pace of changes, and thus bypassing the long and arduous processof gradual changes that the natural course of evolution takes.Techniques used in gene-directed evolution include error-prone PCR, andDNA shuffling technique, etc. For detailed description of thegene-directed evolution method, see Beaudry A A, Joyce G F. Directedevolution of an RNA enzyme. Science, 1992, 257: 641-644.

[0050] Recent development in modern biotechnology provides a variety oftechniques such as error-prone PCR technology, DNA Shuffling, PhageDisplay technique, etc, in order to achieve TSP molecule, or EFGRmolecular vaccine made with gene-directed evolution technique.

[0051] 2. EGFR Recombinant Gene Vaccine

[0052] The present invention can be described at the gene level usingEGFR recombinant gene vaccine, as described below. Gene vaccine, alsoknown mainly as DNA vaccine, is a new type of vaccine based on nucleicacids made with modern molecular technology.

[0053] The EGFR molecular vaccine of the present invention include DNAvaccine. We search for sequences of EGFR collected in publicly availabledatabanks such as GenBank (including gene sequences, cDNA sequences,mRNA sequences and amino acid sequences). Using these sequences, wedesigned primers or probes, and using techniques such PCR, RT-PCR,molecular hybridization, we isolated, from various commercial genelibrary, cDNA library, cell lines, tissue cultures, we cloned andisolated the intracellular domains of the EGFR molecules from a varietyof different species. We have found that the extracellular domain ofEGFR is the preferred active region causing immunogenicity. In addition,we can further use the gene directed evolution technique to select EGFRmolecules with stronger immunogenicity. After confirming the sequencesof the extracellular domain of the EGFR cDNAs using sequencing, weconstructed Eukaryotic plasmid expression systems containing theseintracellular domains of the EGFR using molecular cloning technique.These constructs are then transfected to CHO cell lines, and they areobserved and tested for its EGFR expression and the level of expression.The recombinant EGFR constructs of these Eukaryotic plasmid expressionsystems can be analyzed and confirmed using restriction enzymedigestion, SDS-PAGE, and Western Blot. We used Alkaline Extraction toobtain confirmed recombinant EGFR expression plasmids, and then usingultracentrifuge, ultra-filtration methods to eliminate E. Coliendotoxins. After that, we get pure recombinant plasmid DNA. The plasmidDNA molecules can be used as DNA vaccine for use to immunize animals.

[0054]FIG. 2 shows some representative plasmids used to make the EGFRDNA molecular vaccine.

[0055] A. pORF Based Plasmids:

[0056] The detailed process for constructing these plasmids is describedbriefly as follows: we use public database such as GenBank to obtain thecDNA sequences for human, mouse, Chicken, and they are listed herein asSEQ ID NO 1-5,7-9,19, respectively. We designed the following primerbased upon the cDNA sequences: Human Primer:5′GACCATGGAGGAAAAGAAAGTTTGC3′, 5′ACGAATTCTTAGGACGGGATCTTAGGCCCA3′; MousePrimer: 5′GACCATGGAGGAAAAGAAAGTCTGC3′,5′ACGAATTCTTAATAGATGGTATCTTTGGC3′; Chicken Primer:5′GACCATGGAGGAGAAGAAAGTTTGTC3′, 5′ACGAATTCTTAAGATGGAGTTTTGGAGCC3′.

[0057] We use the total RNA from human lung cancer cell line A431, mouselung cancer cell line LL2, and Chick Embryo to undertake RT-PCRamplification, then collect and purify the amplified EGFR fragment(average 1.9 kb in length) using electrophoresis, and then subclone thePCR products. After confirming the sequences of the PCR subclonesthrough sequencing, we digest them using NcoI and EcORI, collect the 1.9kb fragments and purify them, and then insert the fragments into thepORF-MCS vector (from the InvivoGen Corporation) which had been digestedwith NcoI and EcORI. We select recombiant plasmids. Candidaterecombinant plasmids are confirmed by both restriction enzyme digestionanalysis and PCR amplification. They are named pORF-hEGFR, pORF-mEGFRand pORF-chEGFR.

[0058] B. pcDNA Based Plasmids:

[0059] For the construction of pcDNA-hEGFR pcDNA-mEGFR

pcDNA-chEGFR, the process is similar to the above described process. Inthis case, we used pcDNA3.1(+) vector made by InvitroGen, and vary thesequences of the PCR primers somewhat from those primers described inthe above paragraph. Human Primer: 5′GAGCTAGCATGGAGGAAAAGAAAGTTTGC3′,5′CACTCGAGTTAGGACGGGATCTTAGGCCCA3′; Mouse Primer:5′GAGCTAGCATGGAGGAAAAGAAAGTCTGC3′, 5′CACTCGAGTTAATAGATGGTATCTTTGGC3′;Chicken Primer: 5′GAGCTAGCATGGAGGAGAAGAAAGTTTGTC3′,5′CACTCGAGTTAAGATGGAGTTTTGGAGCC3′.

[0060] We used the DNA vaccine constructed and made with human EGFRgene's extracellular domain, and immunized Lewis Lung Cancer Model Mice.We discovered that 8 weeks after immunization, the survival rate of miceinjected with the EGFR DNA vaccine is 78%, significantly higher thatthose mice that were injected with mouse EGFR DNA vaccine (with asurvival rate of 25%), and much higher than those mice that were used asa control group (with a survival rate of 10-15%). At the same time, wedid not find any pathological changes to the lungs, liver, heart, andkidney of the experimental model mice. Further research was conductedand demonstrated that the induced self-autoimmune response in mice ismainly dependent upon CD4+ T lymphocytes. Testing using the CTL activitytest found no target cells-specific cytocidal effect.Immunohistochemistry results showed deposition of autoantibodies intumor tissues, but no such deposition in non tumor tissues such as lung,liver, etc. The autoantibody in this case is mainly IgG.

[0061] What is disclosed in the present invention is different fromanti-sense RNA and RNAi molecules designed with EGFR molecule astemplate. Those molecules can be viewed as a specific example of theEGFR recombinant gene vaccine. Their mechanism of action is not throughincreasing the immunogenicity of EGFR molecule, thereby inducinganti-EGFR antibody and specific CTL reaction to achieve the goal ofblocking the EGFR signal pathway, thus further inducing tumor cellapoptosis, and inhibiting tumor cells growth and metastasis. Rather,these antisense RNA and RNAi molecules act at the DNA and RNA level bydirectly repressing and prohibiting the expression of EGFR molecules.

[0062] 3. EGFR Recombinant Protein Vaccine

[0063] Protein vaccine is a relatively traditional vaccine, however,protein possess very good immunogenicity. One of the vaccinesencompassed in the present invention is recombinant protein vaccine,including those constructed with various expression systems such as E.coli recombinant expression vectors, yeast recombinant expressionvectors, baculovirus recombinant expression vectors.

[0064] We first prepare the recombinant EGFR constructs as described inSection 2. Namely, using molecular cloning technique such as PCR,RT-PCR, molecular hybridization, we isolated, from various commerciallyavailable gene libraries, cDNA library, cell lines, tissue cultures, wecloned and isolated the intracellular domains of the EGFR molecules froma variety of different species. In addition, we can further use the genedirected evolution technique [describe this technique, using a referenceis ok] to select EGFR molecules with strong immunogenicity. Afterconfirming the sequences of the extracellular domain of the EGFR cDNAsusing sequencing, we constructed prokaryotic plasmid expression systemscontaining these intracellular domains of the EGFR using molecularcloning technique. We transformed suitable E. coli host, observed andexamined their EGFR expression levels. The recombinant EGFR constructscan be analyzed and confirmed using restriction enzyme digestion,SDS-PAGE, and Western Blot. After confirmation, the recombinant EGFRmolecules are used to transform E. coli, the transformed E. coli cellsare grown in a large quantity in order to produce recombinant proteins.Next, we used low temperature ultra centrifuge to harvest thetransformed cells, and then resuspended the cells in PBS solution, andthen lyse the cells using ultrasound techniques. We then isolated andpurified the recombinant EGFR protein using ion-exchange chromatographyand affinity chromatography. The isolated recombinant EGFR protein canbe used as protein vaccine to immunize animals.

[0065] Representative plasmids for recombinant EGFR protein vaccineexpressed in E. coli are shown in FIG. 2B. The detailed process toconstruction these plasmids is described briefly as follows:

[0066] We obtain the cDNA sequences of EGFR from human, mouse, andchicken from public databases such GenBank, corresponding to SEQ ID NO1-5,7-9,19, respectively. Based upon these sequences, we designed thefollowing primers: Human Primer: 5′GACCATGGAGGAAAAGAAAGTTTGC3′,5′ACAGATCTAGGACGGGATCTTAGGCCCA3′; Mouse Primer:5′GACCATGGAGGAAAAGAAAGTCTGC3′, 5′ACAGATCTATAGATGGTATCTTTGGC3′; ChickenPrimer: 5′GACCATGGAGGAGAAGAAAGTTTGTC3′, 5′ACAGATCTAGATGGAGTTTTGGAGCC3′),

[0067] We use pORF-hEGFR, pORF-mEGFR

pORF-chEGFR as template for PCR amplification, then collect and purifythe amplified EGFR fragment (average 1.9 kb in length) usingelectrophoresis, and then subclone the PCR products. After confirmingthe sequences of the PCR subclones through sequencing, we digest themusing NcoI and BgIII, collect the 1.9 kb fragments and purify them, andthen insert the fragments into the pQE60 vector (from the QiagenCorporation) which had been digested with two restriction enzymes NcoIand BgIII. We select recombiant plasmids. Candidate recombinant plasmidsare confirmed by both restriction enzyme digestion analysis and PCRamplification. They are named pQE-hEGFR, pQE-mEGFR

pQE-chEGFR.

[0068] Recombinant EGFR proteins can be produced by methods other thanthe E. coli recombinant expression systems as described above. Forexample, one can use the yeast recombinant expression system,baculovirus recombinant expression system. These recombinant EGFRproteins made from all these systems can be used as recombinant proteinvaccines. See FIG. 2 of the old application for a flow chart of theconstruction process of the EGFR recombinant protein vaccine.

[0069] Respresentative maps for plasmid expressing EGFR recombinantprotein vaccine made from yeast expression system are shown in FIG. 2C.The detailed procedure is described as follows:

[0070] Again, as described in the previous sections, we obtain the cDNAsequences of EGFR from human, mouse, and chicken from public databasessuch GenBank, corresponding to SEQ ID NO 1-5,7-9,19, respectively. Basedupon these sequences, we designed the following primers. Human Primer:5′ATACTCGAGAAAAGAGAGCTGGAGGAAAAGAAAG3′, 5′GCTCTAGAATGGCACAGGTGGCACA3′;Mouse Primer: 5′ATGCTCGAGAAAAGAGAGTTGGAGGAAAAGAAAGTC3′,5′AAGCGGCCGCCATAGATGGTATCT TTG3′; Chicken Primer:5′ATACTCGAGAAAAGAGAGGTGGAGGAGAAGAAAG3′, 5′CGTCTAGAAGATGGAGTTTTGGAG3′

[0071] We use pORF-hEGFR, pORF-mEGFR

pORF-chEGFR as template for PCR amplification, then collect and purifythe amplified EGFR fragment (average 1.9 kb in length) usingelectrophoresis, and then subclone the PCR products. After confirmingthe sequences of the PCR subclones through sequencing, we digest themusing two restriction enzymes XhoI and XbaI (in the case of plasmidscontaining mouse EGFR, we use XhoI and NotI double enzyme digestion),collect the 1.9 kb fragments and purify them, and then insert thefragments into pPICZ A vector (from Invitrogen Corporation) which hadbeen digested with two restriction enzymes XhoI and XbaI (in the case ofplasmids containing mouse EGFR, we use XhoI and NotI double enzymedigestion). We transformed E. coli with these plasmid preparations, andselect recombiant plasmids. Candidate recombinant plasmids are confirmedby both restriction enzyme digestion analysis and PCR amplification.They are named yeast expression plasmids: pYE-hEGFR, pYE-mEGFR andpYE-chEGFR.

[0072] After digesting these yeast expression plasmids with PmeI tolinerize them, we use electric perforation method to transform yeastcell lines X33, or GS115. We use Zeocin resistance to select stabletransformants. We use MMH (Minimal Methanol with histidine, MMH) and MDH(Minimal Dextrose with histidine, MDH) agar plate) to determine andselecte Mut+ transformants. We select 6-10 Mut+ transformants forsmall-scale expression, and then use SDS-PAGE Western Blots ELISA, etc.to confirm the expressed recombinant protein. We select the Mut+transformants with the highest expression efficienty, and culture themat a large scale so as to establish yeast expression seed libraries. Weuse large flasks to culture or ferment the recombinant yeast cell lines,collect the yeast pellets using low temperature centrifuge. Afterresuspend the pellets n PBS solution, we use ultrasound to break thecells. Then, we used Ion Exchange Chromatography and affinitychromatography to isolate and purify the recombinant EGFR protein. Therecombinant EGFR protein derived as such can be used as protein vaccineto immunize subjects. Similarly, yeast recombinant expression plasmidsmade with EGFR can be produced with other yeast expression systems.

[0073] Recombinant protein vaccine has a stronger effect than DNAvaccine in terms of the ability to illicit immune cross-reaction. It canstimulate production of high-titer anti-EGFR antibody and specific CTLactivity thereby can inhibit the growth and metastasis of tumor cells.

[0074] 4. EGFR Recombinant Viral Vaccine

[0075] Recombinant viruses are also a good choice as a system forproducing molecular vaccine. Molecular vaccines made with theserecombinant viral expression systems include recombinant adenovirusvaccine, adenovirus-related viral vaccine, Retroviral viral vaccine,Lenti Virus vaccine, vaccinia virus vaccine, and herpes simplex virusvaccine.

[0076] Currently, Adenovirus vector is one of the most effective vectorsin tumor gene therapy. It has the advantage of having high titer, safe,and can infect dividing, or non-dividing cells, and it does notintegrate into the chromosomes of the host. In addition, adenovirus hasrelatively strong immunogenicity, which is perhaps a downside in genetherapy, but a strong point in gene immunotherapy. As disclosed in thepresent invention, recombinant adenovirus-derived EGFR recombinant viralvaccine is one of the most important embodiments.

[0077] As described in the procedure above, we first cloned variouskinds of autologous, xenogeneic, or gene directed evolutionary EGFRcDNA, and then use these cDNAs to construct recombinant adenoviralexpression vectors, using molecular biological techniques. We thentransfect 293 cells, and harvested the resulting recombinant adenovirus.The recombinant adenovirus is further confirmed using PCR, Western Blot,etc. We took the EGFR recombinant adenoviral vaccine made in largequantity using 293 cells, isolated and purified the recombinant virususing ultracentrifuge, ultra-filtration methods. The EGFR recombinantviral vaccine purified above can be used as vaccine to immunizesubjects. See FIG. 4A for a flow chart showing the construction of EGFRrecombinant adenoviral vaccine. Because adenovirus can introduce geneseffectively, EGFR recombinant viral vaccine made with adenovirus caneffectively induce the anti-tumor immune response in the subject, andthereby inhibiting the growth of tumors with over-expressed EGFR.

[0078] We construct the extracellular domain of EGFR as described inExample One and Two, and then use the AdEasy Systems to constructrecombinant adenovirus in the following manner: we inserted the EGFRsegment into the adenovirus vector's precursor expression plasmidpShuttle-CMV in order to construct the precursor expression vectorpCMV-EGFR. Then, we co-transform E. coli BJ5183 with two components: oneis the PmeI digested adenovirus precursor expression vector pCMV-EGFR,the other is the backbone vector pAdEasy, which contained the adenovirusgenome. After the transformation, we obtained recombinant adenovirusvector plasmid pAd-EGFR. We used the restriction enzyme PacI to digestpAd-EGFR, and use. Calcium Phosphate-DNA precipitation method totransfect the digested pAd-EGFR into adenovirus packaging cell line 293cells, resulting the corresponding recombinant adenovirus Ad-EGFR. Weused PCR, Western Blot and Restriction enzyme digestion analysis toconfirm that the EGFR gene has been indeed incorporated into theadenoviral vector, and that it was expressed effectively in Eukaryoticcells. We used ultracentrifuge to collect large quantity of therecombinant adenovirus Ad-EGFR, and then tested the titer (pfu) of eachbatch of the recombinant virus using upper layer agarose method, andTCID50 method. We then used 293 cells to produce large amount ofconfirmed EGFR adenovirus vaccine. We further isolated and purified therecombinant virus using ultracentrifuge and ultrafiltration. Thepurified EGFR recombinant adenovirus can be used a vaccine to immunizesubject animals.

[0079] As a representative of the EGFR Recombinant Virus Vaccine, wedescribed below the construction of EGFR adenovirus vaccine, which isbased upon the AdEasy system.

[0080] The process is as follows:

[0081] We obtain the cDNA sequences of EGFR from human, mouse, andchicken from public databases such GenBank, corresponding to SEQ ID NO1-5,7-9,19, respectively. Based upon these sequences, we designed thefollowing primers: Human Primer: 5′GAAGATCTATGGAGGAAAAGAAAGTTTGC3′,5′ACGATATCTTAAGGACGGGATCTTAGGCCCA3′; Mouse Primer:5′GAAGATCTATGGAGGAAAAGAAAGTCTGC3′, 5′ACGATATCTTAATAGATGGTATCTTTGGC3′;Chicken Primer: 5′GAAGATCTATGGAGGAGAAGAAAGTTTGTC3′,5′ACGATATCTTAAGATGGAGTTTTGGAGCC3′;

[0082] We use pORF-hEGFR, pORF-mEGFR

pORF-chEGFR as template for PCR amplification, then collect and purifythe amplified EGFR fragment (average 1.9 kb in length) usingelectrophoresis, and then subclone the PCR products. After confirmingthe sequences of the PCR subclones through sequencing, we digest themusing two restriction enzymes BgIII and EcoRV double enzyme digestion,collect the 1.9 kb fragments and purify them, and then insert thefragments into pShuttle-CMV vector (made by Quantum Biotechnologies)which was pre-digested with BgIII and EcoRV. We select recombiantplasmids. The resulting candidate recombinant plasmids are confirmed byboth restriction enzyme digestion analysis and PCR amplification. Theyare named Adenovirus shuttle expression plasmids: pShuttle-hEGFR,pShuttle-mEGFR

pShuttle-chEGFR. See FIG. 2D for plasmid maps of these Adenovirusshuttle expression plasmids.

[0083] We then take the various Adenovirus shuttle expression plasmidsas described before, digest them with PmeI enzyme, and co-transform E.coli BJ5183 cells with backbone vector containing the Adenovirus genomepAdEasy-1 or pAdEast-2. The resulting recombinant Adenovirus vectorplasmids are named pAd-hEGFR1 pAd-mEGFR and pAd-chEGFR. These Adenovirusvector plasmids are digested with PacI enzyme, and then using theCalcium-phosphate-DNA coprecipitation method, they are transfected intothe Adenovirus packaging cell line 293 cells. The resulting recombinantAdenovirus are called Ad-hEGFR, Ad-mEGFR

Ad-chEGFR. PCR, Western blot and other methods are used to confirm thatthe EGFR gene has been indeed integrated into the Adenovirus vector, andthat EGFR has been correctly, and efficiently expressed in Eukaryoticcells.

[0084] Depending upon the difference of the Adenovirus Genome, therecombinant Adenovirus vaccine can be classified into two groups: thefirst group is called the generation I of EGFR Recombinant Adenovirus,resulting from the recombination between Adenovirus Shuttle expressionplasmid pShuttle-EGFR and AdEasy-1 recombinant, thus named Ad-hEGFR IAd-mEGFR I and Ad-chEGFR I. The second group is called the generation IIof EGFR Recombinant Adenovirus, resulting from the recombination betweenAdenovirus Shuttle expression plasmid pShuttle-EGFR and AdEasy-2recombinant, thus named Ad-hEGFR II Ad-mEGFR II and Ad-chEGFR II.

[0085] In addition, the EGFR Recombinant Virus Vaccine can be modifiedspecifically to increase its targeting ability with the compound Mannan.

[0086]Lentivirus vector is a new generation gene thearpy vector. Itoriginated from lentivirus which has a HIV-1 replication deficiency. Itis different from the traditional reverse transcriptase virus vectororiginated from the Moloney Leukemia Virus (MoMLV). The difference isthat Lentivirus vector can effectively transfect both dividing andnon-dividing mammalian cells, and it has good biological safety. Inaddition, Lentivirus vector differs from Adenovirus vector in thatLentivirus vector can integrate into the host cell's chromosomes, andthus enabling the introduced exogenous gene's stable and long-termexpression. Recombinant Lentivirus vector is also an important variationin EGFR recombinant virus vaccine.

[0087] The process to make EGFR Recombinant Lentivirus Vaccine issimilar to what is described with respect to Adenovirus. That is, wefirst clone various autologous, xenogeneic, or gene-directed evolutionEGFR cDNA, and then using molecular biology technique, we construct itsRecombinant Lentivirus expression vector, then transfect 293FT cells,resulting in recombinant Lentiviruses. The recombinant Lentiviruses areconfirmed using PCR, Western Blot, etc. Further, after confirmation, weuse 293FT cells to amplify large quantity of the EGFR RecombinantLentivirus vaccine, and further isolate and purify the recombinantLentivirus using ultracentrifuge and ultrafiltration. The purified EGFRRecombinant Lentivirus can be used as vaccine to immunized subjects. SeeFIG. 4B for a flow chart of the construction of Recombinant Lentivirusvaccine.

[0088] As an illustration of the process of making EGFR RecombinantLentivirus, we use the ViraPower Lentiviral Gateway Expression Kit madeby Invitrogen Corporation. The detailed process is as follows: we firstsearch public database such as GenBank to obatin cDNA sequences of EGFRmolecule of human, mouse and chicken. Their sequences are listed as SEQID NO 1-5,7-9,19, respectively.

[0089] We then designed the following PCR primers: Human Primer:5′GACCATGGAGGAAAAGAAAGTTTGC3′, 5′ACGATATCAGGACGGGATCTTAGGCCCA3′; MousePrimer: 5′GACCATGGAGGAAAAGAAAGTCTGC3′, 5′ACGATATCATAGATGGTATCTTTGGC3′;Chicken Primer: 5′GACCATGGAGGAGAAGAAAGTTTGTC3′,5′ACGATATCAGATGGAGTTTTGGAGCC3′

[0090] We use pORF-hEGFR, pORF-mEGFR

pORF-chEGFR as template for PCR amplification, then collect and purifythe amplified EGFR fragment (average 1.9 kb in length) usingelectrophoresis, and then subclone the PCR products. After confirmingthe sequences of the PCR subclones through sequencing, we digest themusing two restriction enzymes NcoI and EcoRV double enzyme digestion,collect the 1.9 kb fragments and purify them, and then insert thefragments into pENTR11 vector(made by Invitrogen) which was pre-digestedwith NcoI and EcoRV. We select recombiant plasmids. The resultingcandidate recombinant plasmids are confirmed by both restriction enzymedigestion analysis and PCR amplification. They are named as pENTR-hEGFR,pENTR-mEGFR

pENTR-chEGFR. See FIG. 2E for plasmid maps of these Lentivirusexpression plasmids.

[0091] We then take the various Lentivirus expression plasmids asdescribed before, co-transform E. coli DH5 cells together with backbonevector containing the Lentivirus genome pLenti6/V5-DEST. The resultingrecombinant Lentivirus vector plasmids are named pLenti-hEGFR,pLenti-mEGFR and pLenti-chEGFR. See also FIG. 2E. We then mix theseRecombinant Lentivirus vector plasmids with packaging mix, the ViraPowerPackaging Mix, and then using the Calcium-phosphate-DNA coprecipitationmethod, the Recombinant Lentivirus vector plasmids are transfected intothe Lentivirus packaging cell line 293FT cells. The resultingrecombinant Lentivirus are called Lenti-hEGFR, Lenti-mEGFR andLenti-chEGFR. PCR, Western blot and other methods are used to confirmthat the EGFR gene has been indeed integrated into the Lentivirusvector, and that EGFR has been correctly, and efficiently expressed inEukaryotic cells.

[0092] 5. Modification and Improvement of EGFR Molecular Vaccine

[0093] What is described above is a basic procedure of preparingrecombinant tumor associated protein vaccine, and in the preferredembodiment, recombinant EGFR molecular vaccine. We now describe moreembodiments below which include modifications and improvements that canenhance the efficacy and specificity of the EGFR molecular vaccine.

[0094] 1) Choice of Adjuvant for the EGFR Molecular Vaccine

[0095] Suitable adjuvant is carriers for vaccines that can increase theeffective immune response of the vaccine. Different vaccines havedifferent adjuvant. In the present invention, for the EGFR DNA vaccine,we use Freund's adjuvant and liposome. For the EGFR recombinant proteinvaccine, we mainly use aluminum adjuvant, and for the EGFR recombinantviral vaccine, generally, no adjuvant is used.

[0096] 2) EGFR Gene-Modified Vaccine

[0097] Gene-modified vaccine refers to cell vaccine derived from human,other xenogeneic organism's cell lines transfected with EGFR molecule.These cell vaccines include vaccine made using various stablytransformed tumor cell lines, vascular endothelial cells and dendriticcells. They also include vaccines made by using tumor cells or tissuesinfected by recombinant viruses. EGFR gene-modified vaccine is aparticular example of EGFR molecular vaccine.

[0098] As discussed above in the present invention, EGFR is highlyexpressed in a variety of tumors such as lung cancer, breast cancer,ovarian cancer, colorectal cancer, prostate cancer, stomach cancer,bladder cancer, head and neck squamocarcinoma, and glioma, etc. However,EGFR is tolerated in all these tumors and thus cannot elicit immuneresponse. However, by using gene-modified vaccine, we can break theimmune tolerance in these cells, and thus induce anti-EGFR immuneresponse. The procedure of using gene-modified vaccine is similar asdiscussed in the above section. We use Eukaryotic plasmids containingrecombinant EGFR from a variety of different sources to transfectvarious kinds of tumor cell lines such as lung cancer line A431, breastcancer cell line MCF7, etc, or, vascular endothelial cells. Aftertransfection, we select stable transfectants containing stably expressedEGFR molecules. We harvest the transfectants, and generate cellularvaccines using paraformaldehyde fixation method. In addition, we canalso use various EGFR recombinant viral vaccine from different sourcesto infect tumor cell lines, tumor tissue, and tumor vascular endothelialcells, etc. We then can generate cellular vaccine using the sameparaformaldehyde fixation method.

[0099] 3). Stable EGFR Transformants of Commensal Bacteria

[0100] EGFR molecular vaccine also includes live vaccine, which is thestable EGFR transformants of commensal bacteria.

[0101] To produce these live vaccines, we use prokaryotic recombinantexpression plasmids constructed with EGFR from various different sourcessuch as autologous, xenogeneic, and gene-directed evolutionary EGFR. Weuse these plasmids to transform intestinal commensal bacteria such asBifidobacterium. We then select stable transformant. These stable EGFRtransformants of commensal bacteria can continuously secrete exogenousEGFR molecules, thereby inducing the body's immune response, thus theyare categorized as live vaccine. It is a particular example of the EGFRmolecular vaccine.

[0102] 4) Targeted Nano Particles for EGFR Molecular Vaccine

[0103] As shown in the above sections, various embodiments of tumorassociated protein molecular vaccine, such as EGFR molecular vaccine canbe applied to induce effective immune response to inhibit tumor growth.However, these pure recombinant molecular vaccine, such as EGFRmolecular vaccine (including EGFR recombinant gene vaccine, proteinvaccine and viral vaccine) still can be improved to enhance itsspecificity and immunogenicity. The present invention discloses the useof nanotechnology to enhance and modify the EGFR molecular vaccine, byusing targeted nanoparticles in making the EGFR molecular vaccine. Theprotocol includes the following steps:

[0104] We use EGFR molecules, either its DNA or protein form, as targetantigen molecule, we use biological molecules such as liposome,degradable polymer biological materials such as polyDL-lactide-co-glycolide polymer (PLGA) as nano materials, we use MIP-3α

Flt3-L as modifying genes, we use dendritic cells as target cells, withthese materials, we can construct a new type of targeted EGFR vaccinenanoparticles. There are two groups of these nano particles: The firstgroup include nanoparticles made with nano liposomes, or, PLGA, asmaterials to manufacture nanoparticles carrying plasmids which canexpress EGFR in a high efficiency manner. The outside surface of thesenano particles are modified with Flt3-L, or Mannan. The second groupincludes nanoparticles made with liposome, or PLGA, as basic materials,such nanoparticles can carry plasmids with highly expressed MIP-3αgenes, and at the same time, the nano particles also contain otherplasmids highly expressing EGFR molecules.

[0105] EGFR molecular vaccine modified with targeted nano particles canfurther improve effectively the immunogenicity of EGFR, thereby caninduce a stronger anti-tumor immune response, as compared to regularEGFR molecular vaccine without the modification of targeted nanoparticles. See FIG. 4 of the priority application for schematicdescription of targeted nano particles containing EGFR molecularvaccine.

[0106] The targeted nanoparticles in the present invention come in avariety of forms, including

[0107] 1) mannan-modified nanoparticle, including mannan-modifiedrecombinant adenoviral EGFR vaccine and protein vaccine, mannan-modifiedliposome EGFR gene vaccine and protein vaccine),

[0108] 2) gene-targeted nanoparticle, including gene-targetednanoliposome EGFR vaccine, gene-targeted nano-PLGA EGFR vaacine, andgene-targeted adenoviral EGFR vaccine, which express EGFR and MIP-3βsimultaneously,

[0109] 3) recombinant adenoviral EGFR vaccine targeting cancer vescularendothelial cell, including RGD-modified recombinant adenoviral EGFRvaccine)

[0110] As disclosed in the present invention, the diameter of thenanoparticles is generally less than 500 nm, and they can be classifiedinto three sizes: 200-500 nm, 100-200 nm, and 50-100 nm. It ispreferable to use nanoparticles with diameter ranging from 50 nm to 100nm, and the nano peak value at around 80 nm.

[0111] The following procedure applies to the preparation ofMannan-modified Recombinant Adenovirus EGFR vaccine: we first usestandard protocol to obtain and amplify EGFR Recombinant Adenovirus(either Generation I or II), and then use chromatography orultracentrifuge to purify the recombinant adenovirus. Next, we dissolve70 mg Mannan from Sigma into 5 ml 0.1M phosphate buffer (pH6.0) to reacha final concentration of 14 mg/ml, and then add 45 ml 0.01M SodiumPeriodate solution, and mix and oxidize at 4° C. for 60 minutes. Afterthat, we add 10P glycol, and incubate for 30 minutes at 4° C., resultingin Oxidative Mannan (Ox-M) mixture.

[0112] We then load the Ox-M mixture onto Sephadex-G25 columnspreviously balanced with bicarbonate buffer (pH6.0-9.0) and performchromatography, with Ox-M being eluted into 2 ml sized empty vessel.After that, we mix the purified Ox-M with 1×10¹⁴ Recombinant Adenovirusparticles at room temperature overnight, obtaining the neededOx-M-Adenovirus. We then add 1 mg/ml Sodium Borohydride to theOx-M-Adenovirus, leave at room temperature for 3 hours, formingReductive Mannan Adenovirus (Red-M-Adenovirus). Both Ox-M-Adenovirus andRed-M-Adenovirus are desalted by ultrafiltration, and condensed,filtering out bacteria. They are stored in small test tubes, andpreserved at −80° C. The Mannan-modified Recombinant EGFR Adenovirus canbe used as vaccine to immunize a subject.

[0113] As the size of the Adenovirus is around 80 nm, it is considered anatural nanoparticle. Adenovirus particles can be modified so that theAdenoviral fiber protein can express the tri-peptide: RGD. The RGDtripeptide has specific ability to target tumor vascular endothelialcells. Recombinant Adenovirus EGFR vaccine modified with RGD can beviewed as a natural targeted EGFR vaccine nanoparticle.

[0114] In the present invention, we utilize the AdEasy system toconstruct the RDG modified Adenovirus Recombinant EGFR vaccine. Thedetailed process is shown step by step in FIG. 5.

[0115] The detailed process is as follows: we digest the Adenovirusbackbone plasmid pAdEasy-1 and pAdEasy-2 with restriction enzyme SpeI(Sp), and then use T4 DNA Polymerase to fill in the ends (filling, f) soas to make them blunt, and then digest the filled-in product withPacI(P), and recover the 6211 bp and 3579 bp fragments usingelectrophoresis, and name them AdFiber I/Sp/f/P and AdFiber I/Sp/f/P,respectively. These fragments contain the intact Adenovirus fiberprotein gene. Separately, prepare the pSuttle vector by digesting itwith BamHI first, and then fill in with T4 DNA Polymerase, and thendigest with PacI. After such BamHI/filling/PacI-digestion treatment, thevector is ready to be inserted with the AdFiber I/Sp/f/P

AdFiber II/Sp/f/P fragments. The resulting plasmids are namedpSh-AdFiber I and pSh-AdFiber II, respectively.

[0116] We then digest pSh-AdFiber I with NheI enzyme, fill in with T4DNA polymerase, and digest again with KpnI enzyme(NheI/filling/KpnI),recover, using electrophoresis, the 2090 bp fragment called AdFiberI/Nh/f/K; insert this fragment into a pUC 18 vector which had beenpre-digested with SmaI and KpnI double enzyme digestion, resulting inthe recombinant plasmid named pUC-AdFiber I.

[0117] On the other hand, pSh-AdFiber II is digested with AvrII enzyme,then filled in with T4 DNA polyerase, and then digested with HindIII(AvrII/filling/HindIII), using electrophoresis, recover the 838 bpfragment, called AdFiber I/A/f/H. Insert this fragment into a pUC vectorwhich had been previously digested with SmaI and HindIII double enzymedigestion, resulting in new plasmids named pUC-AdFiber II.

[0118] Next, we designed a series of PCR primers so as to usepUC-AdFiber I and pUC-AdFiber II as templates to amplify the Adenovirusknob, (Ad-knob) gene sequences. The primers used are, respectively: F1 (5′GAAAGCTAGC CCTGCAAACATCA3′), R1 ( 5′ACTCCCGGGAGTTGTGTCTCCTGTTTCCTG3′),F2 ( 5′ACTCCCGGGAGTGC ATACTCTATGTCA3′), R2 ( 5′TATGGTACCGGGAGGTGGTGA3′), F3 ( 5′AACCTAGGGAGGTTAACCTAAGCACTG3′), and R3 (5′CTCAAGCTTTTTGG AATTGTTTGA3′).

[0119] Using primer F1-R1, F2-R2, F3-R1 and F2-R3, respectively, for thefirst round PCT, we obtain products PCR1, PCR2, PCR3 and PCR4. Again,using F1-R2 and F3-R3 as primers, and using the amplified products fromthe first round, PCR1 and PCR2, PCR3 and PCR4 as templates, we undertakethe second round of PCR amplification, resulting in PCR productsPCR1-PCR2(PCR I), PCR3-PCR4(PCR II). We take the PCR I and PCR II fromthe amplification in the second round, insert them into pBR322 vectorthat had been previously cut with EcoRV. The resulting recombinantplasmids are named pBR-PCR I and pBR-PCR II.

[0120] The sequence is the RGD-4C duplex is as follows:5′TGTGACTGCCGCGGAGACTGTTTCTGC3′ 3′ACACTGACGGCGCCTCTGACAAAGACG5′

[0121] We insert the RGD-4C into the pBR-PCR I and pBR-PCR II vectors,where the vector were previously digested with SmaI. The resultingrecombinant plasmids are named pBR-PCR/RGD I and pBR-PCR/RGD II. Thesequences of the recombinant plasmids are confirmed using sequencing.Cut pBR-PCR/RGD I with NheI/KpnI double digestion, usingelectrophoresis, rcover the PCR/RGD I fragment. Insert the fragment intothe pUC-AdFiber I vector which had been double digested using NheI/KpnI.The resulting recombinant plasmids are called pUC-AdFiber-RGD I.

[0122] Similarly, we use AvrII/HindIII to double digest pBR-PCR/RGD II,and using electrophoresis, recover the PCR/RGD II fragment. Again,insert the fragment into pUC-AdFiber II vectors previously digested withdouble enzume AvrII/HindIII, the resulting plasmid is namedpUC-AdFiber-RGD II.

[0123] Afterwards, we use SpeI/PacI double enzyme to digestpUC-AdFiber-RGD I and pUC-AdFiber-RGD II vector, and usingelectrophoresis, recover the AdFiber-RGD I, and AdFiber-RGD IIfragments. Insert the fragment into pAdEasy-1, pAdEasy-1 both of whichwere previously digested with SpeI/PacI, the resulting recombinantplasmids are called pAdEasy-RGD I, and pAdEasy-RGD II, respectively.

[0124] Next, we first take pShuttle-hEGFR, pShuttle-mEGFR andpShuttle-chEGFR as described above, and linerize them with PmeI. Afterthat, we co-transform the E. coli BJ5183 cells with these Shuttle-EGFRplasmids together with pAdEasy-RGD I, and pAdEasy-RGD II, respectively.The resulting recombinant plasmids are named Adenovirus plasmidspAd-RGD-EGFR I, and pAd-RGD-EGFR II.

[0125] We used the Adenovirus plasmid pAd-RGD-EGFR I to transfect 394cells, resulting in recombinant Adenovirus named Ad-RGD-EGFR I.Similarly, we transfect Adenovirus plasmid pAd-RGD-EGFR II to 293E4pIXcells, resulting in recombinant Adenovirus named Ad-RGD-EGFR II. Afterpurification, Ad-RGD-EGFR I and Ad-RGD-EGFR II can be used as vaccine toimmunize subjects, and more importantly, these vaccine possessspecificity targeting tumor vascular endothelial cells.

[0126] EGFR molecular vaccine targeted with nanoparticles can increasethe immunogenicity of EGFR greatly compared to normal EGFR molecularvaccine, and thus enabling EGFR to illicit even stronger anti-tumoreimmune response. See FIG. 6 for schematic description of the mechanismof action for targeted EGFR molecular vaccine targeted withnanoparticle.

[0127] 5) Combination of EGFR Molecular Vaccine with Other ImmuneResponse Stimulating Factors

[0128] Combination of EGFR molecular vaccine with other immune responsestimulating factors)

[0129] The anti-tumor effect of EGFR molecular vaccine can be enhancedby the combing effect of other immune response stimulating factors.These factors include cytokines such as IL-2, TNF, IFN-γ, and GM-CSF,etc., chemokines such as MIP3α, MIP3β, and IP10, etc., stringent factorssuch as CEA, HSP70, etc., and various immune co-stimulating factors suchas B7, etc. The combinatorial effect of these immune responsestimulating factors can realize their effects through gene fusion at thegene level, or fusion protein at the protein level. They can also actthrough co-transfecting tumor cells, dendritic cells, and vascularendothelia cells at the cellular level.

[0130] 6. The Anti-Tumor Effect of EGFR Molecular Vaccine

[0131] As discussed above, the present invention provides new type ofmolecular vaccine using tumor associated proteins. As in the specificembodiment of EGFR molecular vaccine, it encompasses protein vaccine,gene vaccine, viral vaccine, and gene-modified vaccine, all made usingautologous, xenogeneic, or gene directed evolutionary EGFR molecules.These molecular vaccines possess effective anti-tumor function includingpreventing tumor formation, inhibit tumor growth, and increase thesurvival rate of human or animals carrying tumors. Its anti-tumormechanism of action is: using EGFR molecule as immune cross-reactionantigen, it can break the body's immune tolerance of self-EGFR molecule,and thus inducing the body's self immune cross reaction against theEGFR. These immune reactions include active immune response includinghumoral and cellular immune response, and passive immune response(adoptive immunity).

[0132] To observe the anti-tumor effect of EGFR recombinant DNA vaccine,mice were randomly divided into groups (15 mice per group), and eachgroup was injected intramuscularly 100 μg EGFR recombinant DNA vaccine,hEe-p, mEe-p, c-p (control plasmid), or Saline, respectively. Theinjection was done once a week for four weeks continuously. One weekafter the forth immunization, immunized mice were each implanted with5×10⁵ of LL/2c Lewis lunger cancer cells (FIGS. 7A and C), or MA782/5Smammary cancer cells (FIGS. 7B and D), respectively. It can be seen fromthe figures that tumors kept growing in mice immunized with mEe-p, c-p,or Saline, while mice immunized with hEe-p exhibited significantprotective immunity. In addition, the survival rate of hEe-p immunizedmice is significantly higher than those of the mice immunized withmEe-p, c-p, or saline. For example, mice immunized with hEe-p livedlonger than 5 months. At 150 days after tumor implantation, miceimmunized with LL/2c Lewis lung cancer and with MA782/5S mammary cancerreached survial rate of 60% and 66%, respectively.

[0133] It is also provided herein that the protective immunity isdosage-dependent. That is, the immunity obtained with dosage of 150%g issimilar to that of 100 μg of the EGFR DNA vaccine, but there is almostno immunity when the dosage is lowered to 5-15 μg. In addition, Micebearing EGFR-negative tumors, such as H22 liver cancer, and MMT-06052mammary cancer exhibited no protective immunity when immunized withhEe-p.

[0134] Other than protective immunity, EGFR recombinant DNA vaccine alsopossesses therapeutic immunity (FIG. 8). As discussed above, mice wererandomly divided into groups of 15. They were each injectedsubcutaneously once weekly for 4 continuous weeks with 1×10 6 LL/2cLewis Lung cancer cells (FIGS. 8A and C), or MA782/5S mammary cancercells (FIGS. 8B and D). Five days later, they were injectedintramuscularly 100 μg each hEe-p, mEe-p, c-p, or saline once weekly for4 continuous weeks. As seen from the figures, tumors in mice immunizedwith mEe-p, c-p, or saline continued to grow, while tumors in miceimmunized with hEe-p exhibited significant therapeutic effect. Inaddition, the survival rate of mice immunized with hEe-p issignificantly higher than those immunized with mEe-p, c-p, or saline.Mice immunized with hEe-p lived longer than 5 months. 150 days aftertumor implantation, mice carrying LL/2c Lewis cancer and MA782/5Smammary cancer has a survival rate of 40% and 53%, respectively.

[0135] EGFR recombinant protein vaccine has similar protective andtherapeutic immunity. See FIG. 9. As described above, 6-8 weeks oldfemale mice which carried C57BL/6 or BALB/c were randomly divided intogroups, and tumor models were established for LL/2c Lewis lung cancer,MA782/5S mammary cancer and C26 intestinal cancer in these mice.Tumor-bearing mice were injected subcutaneously EGFR recombinant proteinvaccine in the amount of 5-50 μg, or alum adjuvant, or saline 100 μg,once a week for 4 weeks continuously. Mice immunized with recombinantchEGFR protein vaccine exhibited significant anti-tumor protectiveimmunity and therapeutic immunity, and the growth of the LL/2c Lewislung cancer, and MA782/5S mammary cancer is inhibited, and the life spanof mice bearing these two tumors is extended, while the EGFR-nativetumor, C26 is not significantly affected. In addition, it can be seenthat tumors kept growing at a fast pace in the control groups where micewere immunized with recombinant mEGFR protein vaccine, alum adjuvant, orsaline, and the life span of these mice were significantly shortened.Comparing to the control group, the tumor volume (t-test) and the lengthof survival (log-rank test) in the test groups showed significantdifference (p<0.05). FIG. 9 shows the anti-tumor effect of EGFRrecombinant protein vaccine in mice bearing MA782/5S mammary cancer.

[0136] Metastasis is a common cause for tumor progression and forfailure of chemothearpy and radiation therapy. The presence of tumorcells in blood and lymph circulation and the formation ofmicrotransferer is key to metastasis. It is disclosed in this inventionthat recombinant EGFR molecular vaccine has anti-metastasis effect ontumor. See FIG. 10. It is discovered during therapy research of LL/2cLewis lung cancer metastasis in animal model by injection in the tailvein, tumor-bearing mice immunized with chEGFR protein vaccine has farless metastasis in lung, or to a much lesser degree comparing to controlgroup, whereas tumor-bearing mice in the control groups where they wereimmunized with recombinant mEGFR protein vaccine, alum adjuvant, orsaline showed 100% metastasis, and they showed countless transferee.

[0137] In addition, EGFR molecular vaccine can inhibit growth of tumorcells in vitro, and they possess adoptive anti-tumor immunity in vivo.See FIG. 11. The process is described as follows: Mice were immunizedwith EGFR DNA vaccine, and sera were collected 7 days after the fourthimmunization. Serum Immunoglobulin (Ig) were purified using affinitychromatography. Various conentration of Ig (1-1000 mg/ml) were added to2×10⁵ EGFR-positive tumor cells (A549, LL/2c, MA782/5S) andEGFR-negative tumor cells (H22 and MMT-06052), both kinds of tumor cellsbeing in log-growth stage. The cells and the Ig were then co-culturedfor 72 hours, and live cells were examined using Tai Pan Blue [spelling]method, and the rate of growth inhibition was calculated. The resultsshowed that EGFR-positive tumor cells that were treated with Ig purifiedfrom the sera of mice immunized with human EGFR recombinant DNA vaccinehEe-p showed significant growth inhibition, whereas EGFR-negative tumorcells were not affected. See FIG. 11A. As a control, Ig derived fromnon-immunized normal mice has no effect on either EGFR-positive tumorcells, or EGFR-negative tumor cells. See FIG. 11B.

[0138] These purified Ig originated from immunized mice also possessadoptive immunity. The process to study adoptive immunity is as follows:nude mice were injected subcutanously 1×10⁵-1×10⁶ tumor cells. One daylater, purified Igs were intravenously injected in the dosage of 10-300mg/kg, twice a week for three weeks continuously. Results showed thatthe adoptive transfer of human EGFR recombinant DNA vaccine hEe-p hassignificant tumor suppression effect. As a control, purified Ig areincubated at 4° C. with fixed EGFR-positive tumor cells or EGFR-nativetumor cells for one hour of shaking and mixing in order for the tumorcells to adsorb the Ig. This process is repeated four times. Resultsshowed that Ig from mice immunized with human EGFR DNA vaccine hEe-pwere pre-adsorbed by the EGFR-positive tumor cells, such as LL/2c andMA782/5S), and thus lost their anti-tumor effect, while the anti-tumoreffect of the Ig still remained after incubating with EGFR-negativetumor cells, such as H22) (FIG. 11C).

[0139] Besides the lung cancer and mammary gland cancer as describedabove, the tumors that can be affected include other solid tumorswherein EGFR is over-expressed, including ovarian cancer, colorectalcancer, prostate cancer, stomach cancer, bladder cancer, head and necksquamocarcinoma and glioma. EGFR molecular vaccine has reliableanti-tumor effect on all cancers mentioned above.

[0140] 7. Mechanism of Action and Biological Safety of the EgfrMolecular Vaccine

[0141] It is well known in modern day immunology that, the mutualrecognition between an antigen or antigenic epitope and itscorresponding receptor/antibody is not merely specific. In fact, thereis a certain degree of plasticity, promiscuity, and degeneracy in thisrecognition. Thus, it is possible to induce immune cross-reactionagainst a self-antigen through the mechanism of molecular mimicry inorder to break the immuno-tolerance for the self-antigen.

[0142] As discussed above, present invention discloses the discoverythat tumor specific proteins such as EGFR molecule is a immunecross-reactive antigen. Certain variation of the EGFR molecule can breakthe body's tolerance for self-EGFR molecule, and induce selfcross-reactive immune response against the EGFR molecule. These immuneresponse against EGFR include active immune response (such as cellularimmune response and humoral immune response), and passive immuneresponse (such as adoptive immune response).

[0143] In order to study the mechanism of the anti-tumor function ofEGFR molecular vaccine, we first undertook immune testing for humoralliquid from EGFR immunized mice. The testing used included flowcytometry, Western Blot, immunoprecipitation, in order to determine theexistence of anti-EGFR autoantibody in mouse and rabbit serum. We usedELISA to test for the titer and type of antibody in mouse/rabbitimmunized sera, we used immunohistochemistry to test the autoantibody inthe tumor tissue of immunized mice. We used tumor cell aggregation test,and serum adoptive immune test for examining the funtion ofautoantibody.

[0144] The following experimental protocols were undertaken. Blood wastaken from each mouse before and after immunization every week usingextraction of blood sample from the mouse tail vein, or after the mouseis sacraficed, sera were collected for use in the next step. Westernblot s shows that antibodies induced by recombinant EGFR molecularvaccine can specifically recognize the corresponding antigen(recombinant EGFR protein or EGFR expression in tumor cells), but theycan not recognize EGFR-negative cells. At the same time, flow cytometrywas used to identify the antibody stereo surface recognition and wefound that recombinant EGFR molecular vaccine can stimulate theproduction of specific antibodies which can in term recognize the EGFRon the surface of tumor cells. Testing for autoantibodies using ELISAshowed that mice started to produce anti-mouse autoantibody two weeksafter immunized with recombinant EGFR molecular vaccine, reaching atiter of 1:100 to 1:5000. It gradualy increased to 1:10000 to 1:500000at week 4, and can maintain a titer of 1:500 to 1:1000 at week 8. As acomparison, the corresponding control group has no detectable antibodyproduction. FIG. 12 showed the types of antibodies produced by miceimmunized with EGFR molecular vaccine (both recombinant DNA vaccine andrecombinant Protein vaccine). From the figure, it can be seen thatrecombinant EGFR molecular vaccine can increase significantly theproduction of IgG1, IgG2a, and IgG2b, but that the production of IgM andIgA is not increased. Moreover, the production of the antibodiesstimulated by recombinant EGFR molecular vaccine can be blocked byanti-CD4 antibody, but not by anti-CD8, anti-NK, or control antibodies.As a control, no specific antibodies were detected in the controlsgroups of protein vaccine, adjuvant, or saline.

[0145] EGFR molecular vaccine can also induce cellular immunity. Thetests used to examine cellular immunity in mice immunized with EGFRmolecular vaccine include: using Cr⁵¹ release method to measure CTLactivities. Using ELISPOT to test cytokine level (mainly for testingserum concentration of IFN-γ, and IL-4), using depletion of immune cellsubsets tests to determine T cell types (CD4+ T lymphocyte, CD8+Tlymphocyte, or NK cells, etc.

[0146] The protocols are carried out as follows: mice are immunized withrecombinant chEGFR vaccine, then spleen monlymphocyte were taken forELISPOT test, which shows there are a large quantity of antigen specificB cells in the monolymphocyte. Testing in mice immunized withrecombinant mEGFR showed the existence of a small amount of antigenspecific B cells. But, no statistically significant amount of antigenspecific B cells were found in control groups including adjuvant groupor blank vector group. In another experiment, spleen T cells were takenfrom mice immunized with recombinant EGFR molecular vaccine for threeweeks, we then use monolymphocyte from health spleen as antigenpresenting cells to activate T cells in vitro in the presence ofantigen. Results show that mice immunized with chEGFR molecular vaccinecontain relatively a larger amount of IFN-γ, and IL-4 producing cells.Spleen T derived from chEGFR molecular vaccine immunized mice cells alsoproduced many IFN-γ, and IL-4 producing cells after being stimulatedagain in vitro by mEGFR molecular vaccine. Both groups of spleen T cellsproduce significantly more than T cells that were not stimulated invitro.

[0147] Furthermore, standard Cr⁵¹ tests were used to determine thespecific cytocidal effect of spleen T cells immunized by recombinantEGFR molecular vaccine against the relevant tumor cells from human andmice. Results showed that after immunized by chEGFR molecular vaccine,at the ratio of 40:1, mouse spleen T cells exhibited specific cytocidalactivity against EGFR positive tumor cells. In the example of LL/2c andMA 782/5S, they are 40.27%, 42.83%, respectively. In contrast, micespleen T cells immunized with mEGFR exhibited no cytocidal effect onLL/2c and MA 782/5S tumor cells, and on EGFR-negative tumor cells suchas C26 cells. At the same time, the cytocidal effect mentioned above canbe blocked by corresponding anti-CD8 and anti MHC-I monoclonalantibodies, but was not blocked by anti-CD4 and anti-MHC-II monoclonalantibodies. Spleen T cells from control groups did not show anystatistically signifcant cytocidal activity.

[0148]FIG. 13 shows the CTL effect of EGFR recombinant DNA vaccine onimmunized mice. It can be seen from this figure that T cells from miceimmunized with human EGFR recombinant DNA vaccine hEe-p has highercytocidal activity against EGFR-positive tumor cells compared to T cellsfrom the control groups. Moreover, this in vitro cytocidal activity canbe blocked by anti-CD8 or anti-MHC-I monoclonal antibody, but not byanti-CD4 monoclonal antibody, showing the this cytocidal activity isdependent on MHC-I dependent CD8+ T cells. In addition, activated spleencells exhibited no increase in NK activity against YAC-1 target cells.Moreover, adoptive transfer of CD4-depleted (CD8+), or CD8-depleted(CD4+) T cells derived from mice immunized with human EGFR recombinantDNA vaccine hEe-p exhibited anti-tumor effect against EGFR-positivetumor. In contrast, its anti-tumor effect is not significant againstEGFR-negative tumor cells. No anti-tumor effect was seen from thecontrol group.

[0149] We also observed long-term potential toxic impact on miceimmunized with EGFR molecular vaccine. No obvious toxic side effectswere observed such as weight loss, skin and hair deterioration, loss ofapetitie, reduced life expectancy. Microscopic examination of the liver,lung, spleen, and brain of immunized mice uncovered no pathologicalchanges. Immunofluorescent staining showed no deposition of autoantibodyin major organs of the mice. See FIG. 14.

EXAMPLES

[0150] The following examples are included for illustrative purposesonly and are not intended to limit the scope of the invention.

Example One EGFR Recombinant DNA Vaccine

[0151] GenBank was searched to obtain the cDNA sequences for human,mouse, Chicken, and they are listed herein as SEQ ID NO 1-5,7-9,19,respectively. We designed the following primer based upon the cDNAsequences:

[0152] For pORF based plasmids: Human Primer:5′GACCATGGAGGAAAAGAAAGTTTGC3′, 5′ACGAATTCTTAGGACGGGATCTTAGGCCCA3′; MousePrimer: 5′GACCATGGAGGAAAAGAAAGTCTGC3′,5′ACGAATTCTTAATAGATGGTATCTTTGGC3′; Chicken Primer:5′GACCATGGAGGAGAAGAAAGTTTGTC3′, 5′ACGAATTCTTAAGATGGAGTTTTGGAGCC3′.

[0153] We use the total RNA from human lung cancer cell line A431, mouselung cancer cell line LL2, and Chick Embryo to undertake RT-PCRamplification, then collect and purify the amplified EGFR fragment(average 1.9 kb in length) using electrophoresis, and then subclone thePCR products. After confirming the sequences of the PCR subclonesthrough sequencing, we digest them using NcoI and EcORI, collect the 1.9kb fragments and purify them, and then insert the fragments into thepORF-MCS vector (from the InvivoGen Corporation) which had been digestedwith NcoI and EcORI. We select recombiant plasmids. Candidaterecombinant plasmids are confirmed by both restriction enzyme digestionanalysis and PCR amplification. They are named pORF-HEGFR, pORF-mEGFRand pORF-chEGFR.

[0154] For pcDNA Based Plasmids:

[0155] For the construction of pcDNA-hEGFR, pcDNA-mEGFR

pcDNA-chEGFR, the process is similar to the above described process. Inthis case, we used pcDNA3.1(+) vector made by InvitroGen, and vary thesequences of the PCR primers somewhat from those primers described inthe above paragraph. Human Primer: 5′GAGCTAGCATGGAGGAAAAGAAAGTTTGC3′,5′CACTCGAGTTAGGACGGGATCTTAGGCCCA3′; Mouse Primer:5′GAGCTAGCATGGAGGAAAAGAAAGTCTGC3′, 5′CACTCGAGTTAATAGATGGTATCTTTGGC3′;Chicken Primer: 5′GAGCTAGCATGGAGGAGAAGAAAGTTTGTC3′,5′CACTCGAGTTAAGATGGAGTTTTGGAGCC3′.

[0156] We selected recombinant expression plasmids, use restrictionenzyme analysis to confirm the presence of the EGFR sequence, and thentransfect them into CHO, NIH3T3, Vero cell lines, respectively. Wemonitor the level of EGFR expression using SDS-PAGE, ELISA, Western Blottechniques. We used alkaline extraction to harvest the recombinant EGFRexpression plasmids. Then, we use ultracentrifuge and ultra-filtrationto eliminate E. coli toxin contamination, and thus obtaining pureplasmid DNA. These plasmid DNAs can be used as DNA vaccine to immunizesubjects, in this case, mice.

[0157] Similar to the above process, we can design other primers orprobes according to the sequences collected in public databases such asGenBank, the sequences including gene, cDNA, mRNA and amino acidsequences. These sequences can be from a variety of species such ashuman, mouse, rat, chicken, mackeral, fruit fly, etc, see SEQ ID Nos:1-14. Using these sequences, we clone and isolate the extracellulardomains of EGFR molecules from different biological sources, using PCR,RT-PCR, molecular hybridization techniques, using commercial genelibraries, cDNA libraries (such as ClonTech, StrateGene's gene librariesand cDNA libraries), and cell lines (such as human lung cancer cell lineA431, mouse Lewis lung cancer line LL2, etc.), or tissues (such as lungcancer, breast cancer tissues, fruit fly tissue, and chick embryo,etc.). Also, we obtained EGFR molecules possessing strong immunogenicityusing selections with the gene-directed evolutionary technique. Afterconfirming the EGFR cDNA extracellular sequences using sequencing, weinserted the EGFR extracellular domain into Eukaryotic expressionplasmids such as pcDNA3.1, pORF-mcs, pBLAST-MCS, pSecTag2, etc.

[0158] We selected mice aged 6-8 weeks old, and established variousmouse tumor models using routine techniques. We injected 100 μg of EGFRrecombinant expression plasmids, once a week, for 4 weeks continuously.We observed the growth of the tumor-bearing mice, and the progression ofthe tumor. After 8 weeks, the mice were sacrificed, and sera of theimmunized mice and organs were collected. We used flowcytometry, ELISA,and Western Blot to test for humoral immune response. To examinecellular response, we used Cr⁵¹, ELISpot methods. To determine toxicityand side effects of the experiments, we used immunohistochemistry. Next,we purified the sera of the immunized mice, used routine methods toestablish tumor models in nude mice, and undertake adoptiveimmunotherapy, and further observe the growth of the tumors.

Example Two EGFR Recombinant Protein Vaccine (Expressed in E. coli)

[0159] As in Example One, we obtain the cDNA sequences of EGFR fromhuman, mouse, and chicken from public databases such GenBank,corresponding to SEQ ID NO 1-5,7-9,19, respectively. Based upon thesesequences, we designed the following primers: Human Primer:5′GACCATGGAGGAAAAGAAAGTTTGC3′, 5′ACAGATCTAGGACGGGATCTTAGGCCCA3′. MousePrimer: 5′GACCATGGAGGAAAAGAAAGTCTGC3′, 5′ACAGATCTATAGATGGTATCTTTGGC3′.Chicken Primer: 5′GACCATGGAGGAGAAGAAAGTTTGTC3′,5′ACAGATCTAGATGGAGTTTTGGAGCC3′.

[0160] We use pORF-hEGFR, pORF-mEGFR

pORF-chEGFR as template for PCR amplification, then collect and purifythe amplified EGFR fragment (average 1.9 kb in length) usingelectrophoresis, and then subclone the PCR products. After confirmingthe sequences of the PCR subclones through sequencing, we digest themusing NcoI and BgIII, collect the 1.9 kb fragments and purify them, andthen insert the fragments into the pQE60 vector (from the QiagenCorporation) which had been digested with two restriction enzymes NcoIand BgIII. We select recombiant plasmids. Candidate recombinant plasmidsare confirmed by both restriction enzyme digestion analysis and PCRamplification. They are named pQE-hEGFR, pQE-mEGFR

pQE-chEGFR.

[0161] Similarly, depending upon different prokaryotic expressionvectors such as pET32, pLLp, pSE420, different primers can be designedand used to construct other EGFR recombinant prokaryotic expressionplasmids.

[0162] Similar to Example One, we cloned the extracellular domain ofvarious EGFR molecules, and then insert the domain into the prokaryoticexpression plasmids such as pQE, pET32, pLLp, etc. After selectingrecombinant expression plasmids, we confirmed the sequences of thedomain using restriction enzyme digestion. We then transformed theserecombinant plasmids into E. coli hosts such as E. coli TOP10F′, E. coliBL21(DE3)pLys, E. coli M15, etc., and then observed and tested the levelof expression of EGFR molecule using techniques such as SDS-PAGE, ELISA,Western Blot, etc. From these testing, we determined the optimal E. colihost for expression the recombinant plasmids. We then used confirmedEGFR expression plasmids to transform the optimal E. coli host cells,and then established stable transformants and various levels of

[spelling] libraries. We cultured the recombinant bacterial cells inshaking incubators, or alternatively, we fermented the bacteria usingstandard methods. We harvested the recombinant bacteria using lowtemperature centrifuge. Then we suspended the bacteria in PBS, and thenbroke the cells using ultrasound method. We used ionic exchangechromatography and affinity chromatography to isolate and purify therecombinant EGFR proteins. The purified EGFR proteins can be used asprotein vaccine to immunize subject mice.

[0163] As in Example One, we selected mice aged 6-8 weeks old, andestablished various mouse tumor models using routine techniques. Weinjected 5-50 μg of EGFR recombinant protein, once a week, for 4 weekscontinuously. We observed the growth of the tumor-carrying mice, and thedevelopment of the tumor. After 8 weeks, the mice were sacrificed, andsera of the immunized mice and organs were collected. We usedflowcytometry, ELISA, and Western Blot to test for humoral immuneresponse. To examine cellular response, we used Cr⁵¹ ELISpot methods. Todetermine toxicity and side effects of the experiments, we usedimmunohistochemistry. Next, we purified the sera of the immunized mice,used routine methods to establish tumor models in nude mice, andundertake adoptive immunotherapy, and further observe the growth of thetumors.

Example Three EGFR Recombinant Protein Vaccine (Expressed in YeastPichia pastoris)

[0164] Again, as described in the previous sections, we obtain the cDNAsequences of EGFR from human, mouse, and chicken from public databasessuch GenBank, corresponding to SEQ ID NO 1-5,7-9,19, respectively. Basedupon these sequences, we designed the following primers. Human Primer:5′ATACTCGAGAAAAGAGAGCTGGAGGAAAAGAAAG3′, 5′GCTCTAGAATGGCACAGGTGGCACA3′;Mouse Primer: 5′ATGCTCGAGAAAAGAGAGTTGGAGGAAAAGAAAGTC3′,5′AAGCGGCCGCCATAGATGGTATCTTTG3′; Chicken Primer:5′ATACTCGAGAAAAGAGAGGTGGAGGAGAAGAAAG3′, 5′CGTCTAGAAGATGGAGTTTTGGAG3′

[0165] We use pORF-hEGFR, pORF-mEGFR and pORF-chEGFR as template for PCRamplification, then collect and purify the amplified EGFR fragment(average 1.9 kb in length) using electrophoresis, and then subclone thePCR products. After confirming the sequences of the PCR subclonesthrough sequencing, we digest them using two restriction enzymes XhoIand XbaI (in the case of plasmids containing mouse EGFR, we use XhoI andNotI double enzyme digestion), collect the 1.9 kb fragments and purifythem, and then insert the fragments into pPICZ A vector (from InvitrogenCorporation) which had been digested with two restriction enzymes XhoIand XbaI (in the case of plasmids containing mouse EGFR, we use XhoI andNotI double enzyme digestion). We transformed E. coli with these plasmidpreparations, and select recombiant plasmids. Candidate recombinantplasmids are confirmed by both restriction enzyme digestion analysis andPCR amplification. They are named yeast expression plasmids: pYE-hEGFR,pYE-mEGFR and pYE-chEGFR.

[0166] After digesting these yeast expression plasmids with PmeI tolinerize them, we use electric perforation method [spelling] totransform yeast cell lines X33, or GS115. We use Zeocin resistance toselect stable transformants. We use MMH (Minimal Methanol withhistidine, MMH)and MDH (Minimal Dextrose with histidine, MDH) agarplate) to determine and selecte Mut+transformants. We select 6-10Mut+transformants for small-scale expression, and then use SDS-PAGE,Western Blot, ELISA, etc. to confirm the expressed recombinant protein.We select the Mut+transformants with the highest expression efficienty,and culture them at a large scale so as to establish yeast expressionseed libraries. We use large flasks to culture or ferment therecombinant yeast cell lines, collect the yeast pellets using lowtemperature centrifuge. After resuspend the pellets n PBS solution, weuse ultrasound to break the cells. Then, we used Ion ExchangeChromatography and affinity chromatography to isolate and purify therecombinant EGFR protein. The recombinant EGFR protein derived as suchcan be used as protein vaccine to immunize subjects. Similarly, yeastrecombinant expression plasmids made with EGFR can be produced withother yeast expression systems.

[0167] As in Example Two, we selected mice aged 6-8 weeks old, andestablished various mouse tumor models using routine techniques. Weinjected 5-50 μg EGFR recombinant protein, once a week, for 4 weekscontinuously. We observed the growth of the tumor-carrying mice, and thedevelopment of the tumor. After 8 weeks, the mice were sacrificed, andsera of the immunized mice and organs were collected. We usedflowcytometry, ELISA, and Western Blot to test for humoral immuneresponse. To examine cellular response, we used Cr⁵¹, ELISpot methods.To determine toxicity and side effects of the experiments, we usedimmunohistochemistry. Next, we purified the sera of the immunized mice,used routine methods to establish tumor models in nude mice, andundertake adoptive immunotherapy, and further observe the growth of thetumors.

Example Four EGFR Recombinant Adenovirus Vaccine

[0168] As in the previous examples, we obtained the cDNA sequences ofEGFR from human, mouse, and chicken from public databases such GenBank,corresponding to SEQ ID NO 1-5,7-9,19, respectively. Based upon thesesequences, we designed the following primers: Human Primer:5′GAAGATCTATGGAGGAAAAGAAAGTTTGC3′, 5′ACGATATCTTAAGGACGGGATCTTAGGCCCA3′;Mouse Primer: 5′GAAGATCTATGGAGGAAAAGAAAGTCTGC3′,5′ACGATATCTTAATAGATGGTATCTTTGGC3′; Chicken Primer:5′GAAGATCTATGGAGGAGAAGAAAGTTTGTC3′, 5′ACGATATCTTAAGATGGAGTTTTGGAGCC3′;

[0169] We use pORF-hEGFR, pORF-mEGFR and pORF-chEGFR as template for PCRamplification, then collect and purify the amplified EGFR fragment(average 1.9 kb in length) using electrophoresis, and then subclone thePCR products. After confirming the sequences of the PCR subclonesthrough sequencing, we digest them using two restriction enzymes BgIIIand EcoRV double enzyme digestion, collect the 1.9 kb fragments andpurify them, and then insert the fragments into pShuttle-CMV vector(made by Quantum Biotechnologies) which was pre-digested with BgIII andEcoRV. We select recombiant plasmids. The resulting candidaterecombinant plasmids are confirmed by both restriction enzyme digestionanalysis and PCR amplification. They are named Adenovirus shuttleexpression plasmids: pShuttle-hEGFR, pShuttle-mEGFR and pShuttle-chEGFR.See FIG. 2D for plasmid maps of these Adenovirus shuttle expressionplasmids.

[0170] We then take the various Adenovirus shuttle expression plasmidsas described before, digest them with PmeI enzyme, and co-transform E.coli BJ5183 cells with backbone vector containing the Adenovirus genomepAdEasy-1 or pAdEast-2. The resulting recombinant Adenovirus vectorplasmids are named pAd-hEGFR, pAd-mEGFR and s5 pAd-chEGFR. TheseAdenovirus vector plasmids are digested with PacI enzyme, and then usingthe Calcium-phosphate-DNA coprecipitation method, they are transfectedinto the Adenovirus packaging cell line 293 cells. The resultingrecombinant Adenoviruses are called Ad-hEGFR, Ad-mEGFR and Ad-chEGFR.PCR, Western blot and other methods are used to confirm that the EGFRgene has been indeed integrated into the Adenovirus vector, and thatEGFR has been correctly, and efficiently expressed in Eukaryotic cells.

[0171] Depending upon the difference of the Adenovirus Genome, therecombinant Adenovirus vaccine can be classified into two groups: thefirst group is called Generation I of EGFR Recombinant Adenovirus,resulting from the recombination between Adenovirus Shuttle expressionplasmid pShuttle-EGFR and AdEasy-1 recombinant, thus named Ad-hEGFR I,Ad-mEGFR I and Ad-chEGFR I. The second group is called Generation II ofEGFR Recombinant Adenovirus, resulting from the recombination betweenAdenovirus Shuttle expression plasmid pShuttle-EGFR and AdEasy-2recombinant, thus named Ad-hEGFR II, Ad-mEGFR II and Ad-chEGFR II.

[0172] We used ultracentrifuge to collect large quantity of therecombinant adenovirus Ad-EGFR, and then tested the titer (pfu) of eachbatch of the recombinant virus using upper layer agarose method, andTCID50 method. We then used 293 cells to produce large amount ofconfirmed EGFR adenovirus vaccine. We further isolated and purified therecombinant virus using ultracentrifuge and ultrafiltration. Thepurified EGFR recombinant adenovirus can be used a vaccine to immunizesubject animals.

[0173] Similar to what's described in the previous examples, we selectedmice aged 6-8 weeks old, and established various mouse tumor modelsusing routine techniques. We injected 1×10⁹ PFU EGFR recombinantadenovirus in each mouse, once a week, for 4 weeks continuously. Weobserved the growth of the tumor-carrying mice, and the development ofthe tumor. After 8 weeks, the mice were sacrificed, and sera of theimmunized mice and organs were collected. We used flowcytometry, ELISA,and Western Blot to test for humoral immune response. To examinecellular response, we used Cr⁵¹, ELISpot methods. To determine toxicityand side effects of the experiments, we used immunohistochemistry. Next,we purified the sera of the immunized mice, used routine methods toestablish tumor models in nude mice, and undertake adoptiveimmunotherapy, and further observe the growth of the tumors.

Example Five EGFR Recombinant Lentivirus Vaccine

[0174] As an illustration of the process of making EGFR RecombinantLentivirus, we use the ViraPower Lentiviral Gateway Expression Kit madeby Invitrogen Corporation. The detailed process is as follows: we firstsearch public database such as GenBank to obatin cDNA sequences of EGFRmolecule of human, mouse and chicken. Their sequences are listed as SEQID NO 1-5,7-9,19, respectively.

[0175] We then designed the following PCR primers: Human Primer:5′GACCATGGAGGAAAAGAAAGTTTGC3′, 5′ACGATATCAGGACGGGATCTTAGGCCCA3′; MousePrimer: 5′GACCATGGAGGAAAAGAAAGTCTGC3′, 5′ACGATATCATAGATGGTATCTTTGGC3′;Chicken Primer: 5′GACCATGGAGGAGAAGAAAGTTTGTC3′,5′ACGATATCAGATGGAGTTTTGGAGCC3′

[0176] We use pORF-hEGFR, pORF-mEGFR and pORF-chEGFR as template for PCRamplification, then collect and purify the amplified EGFR fragment(average 1.9 kb in length) using electrophoresis, and then subclone thePCR products. After confirming the sequences of the PCR subclonesthrough sequencing, we digest them using two restriction enzymes NcoIand EcoRV double enzyme digestion, collect the 1.9 kb fragments andpurify them, and then insert the fragments into pENTR11 vector(made byInvitrogen) which was pre-digested with NcoI and EcoRV. We selectrecombiant plasmids. The resulting candidate recombinant plasmids areconfirmed by both restriction enzyme digestion analysis and PCRamplification. They are named as pENTR-hEGFR, pENTR-mEGFR andpENTR-chEGFR. See. FIG. 2E for plasmid maps of these Lentivirusexpression plasmids.

[0177] We then take the various Lentivirus expression plasmids asdescribed before, co-transform E. coli DH5 cells together with backbonevector containing the Lentivirus genome pLenti6/V5-DEST. The resultingrecombinant Lentivirus vector plasmids are named pLenti-hEGFR,pLenti-mEGFR and pLenti-chEGFR. See also FIG. 2E. We then mix theseRecombinant Lentivirus vector plasmids with packaging mix, the ViraPowerPackaging Mix, and then using the Calcium-phosphate-DNA coprecipitationmethod, the Recombinant Lentivirus vector plasmids are transfected intothe Lentivirus packaging cell line 293FT cells. The resultingrecombinant Lentivirus are called Lenti-hEGFR, Lenti-mEGFR andLenti-chEGFR. PCR, Western blot and other methods are used to confirmthat the EGFR gene has been indeed integrated into the Lentivirusvector, and that EGFR has been correctly, and efficiently expressed inEukaryotic cells.

[0178] We used ultracentrifuge to collect large quantity of therecombinant adenovirus Ad-EGFR, and then tested the titer (pfu) of eachbatch of the recombinant virus using upper layer agarose method, andTCID50 method. We then used 293 cells to produce large amount ofconfirmed EGFR adenovirus vaccine. We further isolated and purified therecombinant virus using ultracentrifuge and ultrafiltration. Thepurified EGFR recombinant adenovirus can be used a vaccine to immunizesubject animals.

[0179] Similar to what's described in the previous examples, we selectedmice aged 6-8 weeks old, and established various mouse tumor modelsusing routine techniques. We injected 1×10⁹ PFU EGFR recombinantLentivirus in each mouse, once a week, for 4 weeks continuously. Weobserved the growth of the tumor-carrying mice, and the development ofthe tumor. After 8 weeks, the mice were sacrificed, and sera of theimmunized mice and organs were collected. We used flowcytometry, ELISA,and Western Blot to test for humoral immune response. To examinecellular response, we used Cr⁵¹, ELISpot methods. To determine toxicityand side effects of the experiments, we used immunohistochemistry. Next,we purified the sera of the immunized mice, used routine methods toestablish tumor models in nude mice, and undertake adoptiveimmunotherapy, and further observe the growth of the tumors.

Example Six Mannan-modified Recombinant Adenovirus EGFR Vaccine

[0180] The following procedure applies to the preparation ofMannan-modified Recombinant Adenovirus EGFR vaccine: we first usestandard protocol to obtain and amplify EGFR Recombinant Adenovirus(either Generation I or II), and then use chromatography orultracentrifuge to purify the recombinant adenovirus. Next, we dissolve70 mg Mannan from Sigma into 5 ml 0.1M phosphate buffer (pH6.0) to reacha final concentration of 14 mg/ml, and then add 45 ml 0.01M SodiumPeriodate solution, and mix and oxidize at 4° C. for 60 minutes. Afterthat, we add 10 μl glycol, and incubate for 30 minutes at 4° C.,resulting in Oxidative Mannan (Ox-M) mixture.

[0181] We then load the Ox-M mixture onto Sephadex-G25 columnspreviously balanced with bicarbonate buffer (pH6.0-9.0) and performchromatography, with Ox-M being eluted into 2 ml sized empty vessel.After that, we mix the purified Ox-M with 1×10¹⁴ Recombinant Adenovirusparticles at room temperature overnight, obtaining the neededOx-M-Adenovirus. We then add 1 mg/ml Sodium Borohydride to theOx-M-Adenovirus, leave at room temperature for 3 hours, formingReductive Mannan Adenovirus (Red-M-Adenovirus). Both Ox-M-Adenovirus andRed-M-Adenovirus are desalted by ultrafiltration, and condensed,filtering out bacteria. They are stored in small test tubes, andpreserved at −80° C. The Mannan-modified Recombinant EGFR Adenovirus canbe used as vaccine to immunize a subject.

[0182] Similar to what's described in the previous examples, we selectedmice aged 6-8 weeks old, and established various mouse tumor modelsusing routine techniques. We injected 1×10¹⁰ PFU EGFR recombinantLentivirus in each mouse, once a week, for 4 weeks continuously. Weobserved the growth of the tumor-carrying mice, and the development ofthe tumor. After 8 weeks, the mice were sacrificed, and sera of theimmunized mice and organs were collected. We used flowcytometry, ELISA,and Western Blot to test for humoral immune response. To examinecellular response, we used Cr⁵¹, ELISpot methods. To determine toxicityand side effects of the experiments, we used immunohistochemistry. Next,we purified the sera of the immunized mice, used routine methods toestablish tumor models in nude mice, and undertake adoptiveimmunotherapy, and further observe the growth of the tumors.

Example Seven RGD-modified Recombinant Adenovirus EGFR Vaccine

[0183] In the present invention, we utilize the AdEasy system toconstruct the RDG modified Adenovirus Recombinant EGFR vaccine. Thedetailed process is shown step by step in FIG. 5.

[0184] The detailed process is as follows: we digest the Adenovirusbackbone plasmid pAdEasy-1 and pAdEasy-2 with restriction enzyme SpeI(Sp), and then use T4 DNA Polymerase to fill in the ends (filling, f) soas to make them blunt, and then digest the filled-in product withPacI(P), and recover the 621 lbp and 3579 bp fragments usingelectrophoresis, and name them AdFiber I/Sp/f/P and AdFiber II/Sp/f/P,respectively. These fragments contain the intact Adenovirus fiberprotein gene. Separately, prepare the 15 pSuttle vector by digesting itwith BamHI first, and then fill in with T4 DNA Polymerase, and thendigest with PacI. After such BamHI/filling/PacI-digestion treatment, thevector is ready to be inserted with the AdFiber I/Sp/f/P

AdFiber II/Sp/f/P fragments. The resulting plasmids are namedpSh-AdFiber I and pSh-AdFiber II, respectively.

[0185] We then digest pSh-AdFiber I with NheI enzyme, fill in with T4DNA polymerase, and digest again with KpnI enzyme(NheI/filling/KpnI),recover, using electrophoresis, the 2090 bp fragment called AdFiberI/Nh/f/K; insert this fragment into a pUC 18 vector which had beenpre-digested with SmaI and KpnI double enzyme digestion, resulting inthe recombinant plasmid named pUC-AdFiber I.

[0186] On the other hand, pSh-AdFiber II is digested with AvrII enzyme,then filled in with T4 DNA polymerase, and then digested with HindIII(AvrII/filling/HindIII), using electrophoresis, recover the 838 bpfragment, called AdFiber I/A/f/H. Insert this fragment into a pUC vectorwhich had been previously digested with SmaI and HindIII double enzymedigestion, resulting in new plasmids named pUC-AdFiber II.

[0187] Next, we designed a series of PCR primers so as to usepUC-AdFiber I and pUC-AdFiber II as templates to amplify the Adenovirusknob, (Ad-knob) gene sequences. The primers used are, respectively:F1(5′GAAAGCTAGCCCTGCAAACATCA3′), R1(5′ACTCCCGGGAGTTGTGTCTCCTGTTTCCTG3′),F2(5′ACTCCCGGGAGTGCATACTCTATGTCA3′), R2(5′TATGGTACCGGGAGGTGGTGA3′),F3(5′AACCTAGGGAGGTTAACCTAAGCACTG3′), andR3(5′CTCAAGCTTTTTGGAATTGTTTGA3′).

[0188] Using primer F1-R1, F2-R2, F3-R1 and F2-R3, respectively, for thefirst round PCT, we obtain products PCR1, PCR2, PCR3 and PCR4. Again,using F1-R2 and F3-R3 as primers, and using the amplified products fromthe first round, PCR1 and PCR2, PCR3 and PCR4 as templates, we undertakethe second round of PCR amplification, resulting in PCR productsPCR1-PCR2(PCR I), PCR3-PCR4(PCR II). We take the PCR I and PCR II fromthe amplification in the second round, insert them into pBR322 vectorthat had been previously cut with EcoRV. The resulting recombinantplasmids are named pBR-PCR I and pBR-PCR II.

[0189] The sequence of the RGD-4C duplex is as follows:5′TGTGACTGCCGCGGAGACTGTTTCTGC3′ 3′ACACTGACGGCGCCTCTGACAAAGACG5′

[0190] We insert the RGD-4C into the pBR-PCR I and pBR-PCR II vectors,where the vector were previously digested with SmaI. The resultingrecombinant plasmids are named pBR-PCR/RGD I and pBR-PCR/RGD II. Thesequences of the recombinant plasmids are confirmed using sequencing.Cut pBR-PCR/RGD I with NheI/KpnI double digestion, usingelectrophoresis, rcover the PCR/RGD I fragment. Insert the fragment intothe pUC-AdFiber I vector which had been double digested using NheI/KpnI.The resulting recombinant plasmids are called pUC-AdFiber-RGD I.

[0191] Similarly, we use AvrII/HindIII to double digest pBR-PCR/RGD II,and using electrophoresis, recover the PCR/RGD II fragment. Again,insert the fragment into pUC-AdFiber II vectors previously digested withdouble enzume AvrII/HindIII, the resulting plasmid is namedpUC-AdFiber-RGD II.

[0192] Afterwards, we use SpeI/PacI double enzyme to digestpUC-AdFiber-RGD I and pUC-AdFiber-RGD II vector, and usingelectrophoresis, recover the AdFiber-RGD I, and AdFiber-RGD IIfragments. Insert the fragment into pAdEasy-1, pAdEasy-1 both of whichwere previously digested with SpeI/PacI, the resulting recombinantplasmids are called pAdEasy-RGD I, and pAdEasy-RGD II, respectively.

[0193] Next, we first take pShuttle-hEGFR, pShuttle-mEGFR andpShuttle-chEGFR as described above, and linerize them with PmeI. Afterthat, we co-transform the E. coli BJ5183 cells with these Shuttle-EGFRplasmids together with pAdEasy-RGD I, and pAdEasy-RGD II, respectively.The resulting recombinant plasmids are named Adenovirus plasmidspAd-RGD-EGFR 1, and pAd-RGD-EGFR II.

[0194] We used the Adenovirus plasmid pAd-RGD-EGFR I to transfect 394cells, resulting in recombinant Adenovirus named Ad-RGD-EGFR I.Similarly, we transfect Adenovirus plasmid pAd-RGD-EGFR II to 293E4pIXcells, resulting in recombinant Adenovirus named Ad-RGD-EGFR II. Afterpurification, Ad-RGD-EGFR I and Ad-RGD-EGFR II can be used as vaccine toimmunize subjects, and more importantly, these vaccines have specificitytargeting tumor vascular endothelial cells.

Example Eight Pharmacological Studies of Recombinant EGFR MolecularVaccine

[0195] It is important to obtain pharmacological studies relating to theanti-tumor effect of recombinant EGFR molecular vaccine. Studies can beperformed for this purpose in the present invention include: ordinarytumor-bearing mice (including protective immunity study, therapeuticimmunity study, and metastasis model study), rabbit immunological test,monkey immunological test, nude mice adoptive immunological test,dosage-dependency test, and in vitro cellular test, etc. In thisexample, we describe the ordinary tumor-bearing mice test, using lungcancer as a model. Other tests can be performed similarly.

[0196] 1. Protective Immunity of Tumor-Bearing Mice:

[0197] 6-8 weeks old C57BL/6 mice are randomly divided into groups. Theyare injected EGFR molecular vaccine in both hind legs intramuscularly,once a week for four weeks continuously. At the 5^(th) week, mice wereare challenged with LL/2c tumor cells in the concentration of 1×10⁶.Blood is taken from tail vein or from sacraficing the mice at week 0, 1,3, 5 after initial immunization. After centrifuge (5000 RPM for 3minutes), sera is collected and stored at −20° C. for future use.

[0198] 2. Therapeutic Immunity of Tumor-Bearing Mice:

[0199] 6-8 weeks old C57BL/6 mice are randomly divided into groups. Theyare challenged with LL/2 cancer cells at a concentration of 1×10⁶. Fourdays after tumor introduction, they are randomly divided into groups,and immunized with EGFR molecular vaccine by injection intramuscularlyin the hind legs, once a week for four weeks continuously. At week 0, 2.4, and 6 after initial immunization, blood is taken from tail vein orfrom sacraficing the mice. After centrifuge (5000 RPM for 3 minutes),sera is collected and stored at −20° C. for future use. Record tumorweight, volume and survial curves.

[0200] 3. Metastasis Model Test of Tumor-Bearing Mice:

[0201] 6-8 weeks old C57BL/6 female mice are randomly divided intogroups. They are injected EGFR molecular vaccine in both hind legsintramuscularly, once a week for four weeks continuously. After twoweeks, LL/2 tumor cells at log growth period are selected, and injectedintramuscularly into the hind legs of the mice, at a concentration of2×10⁵ per mouse. Contiue the injection mentioned above. After fourweeks, mice are killed, and their lungs are weighed. After examinationfor tumor transferer in the lung, the lungs are fixated with 10%neutrally buffered formaldehyde.

[0202] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims.

1 27 1 3633 DNA EE(homo sapiens) CDS (1)..(3630) 1 atg cga ccc tcc gggacg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48 Met Arg Pro Ser Gly ThrAla Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 gcg ctc tgc ccg gcgagt cgg gct ctg gag gaa aag aaa gtt tgc caa 96 Ala Leu Cys Pro Ala SerArg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 ggc acg agt aac aag ctcacg cag ttg ggc act ttt gaa gat cat ttt 144 Gly Thr Ser Asn Lys Leu ThrGln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 ctc agc ctc cag agg atg ttcaat aac tgt gag gtg gtc ctt ggg aat 192 Leu Ser Leu Gln Arg Met Phe AsnAsn Cys Glu Val Val Leu Gly Asn 50 55 60 ttg gaa att acc tat gtg cag aggaat tat gat ctt tcc ttc tta aag 240 Leu Glu Ile Thr Tyr Val Gln Arg AsnTyr Asp Leu Ser Phe Leu Lys 65 70 75 80 acc atc cag gag gtg gct ggt tatgtc ctc att gcc ctc aac aca gtg 288 Thr Ile Gln Glu Val Ala Gly Tyr ValLeu Ile Ala Leu Asn Thr Val 85 90 95 gag cga att cct ttg gaa aac ctg cagatc atc aga gga aat atg tac 336 Glu Arg Ile Pro Leu Glu Asn Leu Gln IleIle Arg Gly Asn Met Tyr 100 105 110 tac gaa aat tcc tat gcc tta gca gtctta tct aac tat gat gca aat 384 Tyr Glu Asn Ser Tyr Ala Leu Ala Val LeuSer Asn Tyr Asp Ala Asn 115 120 125 aaa acc gga ctg aag gag ctg ccc atgaga aat tta cag gaa atc ctg 432 Lys Thr Gly Leu Lys Glu Leu Pro Met ArgAsn Leu Gln Glu Ile Leu 130 135 140 cat ggc gcc gtg cgg ttc agc aac aaccct gcc ctg tgc aac gtg gag 480 His Gly Ala Val Arg Phe Ser Asn Asn ProAla Leu Cys Asn Val Glu 145 150 155 160 agc atc cag tgg cgg gac ata gtcagc agt gac ttt ctc agc aac atg 528 Ser Ile Gln Trp Arg Asp Ile Val SerSer Asp Phe Leu Ser Asn Met 165 170 175 tcg atg gac ttc cag aac cac ctgggc agc tgc caa aag tgt gat cca 576 Ser Met Asp Phe Gln Asn His Leu GlySer Cys Gln Lys Cys Asp Pro 180 185 190 agc tgt ccc aat ggg agc tgc tggggt gca gga gag gag aac tgc cag 624 Ser Cys Pro Asn Gly Ser Cys Trp GlyAla Gly Glu Glu Asn Cys Gln 195 200 205 aaa ctg acc aaa atc atc tgt gcccag cag tgc tcc ggg cgc tgc cgt 672 Lys Leu Thr Lys Ile Ile Cys Ala GlnGln Cys Ser Gly Arg Cys Arg 210 215 220 ggc aag tcc ccc agt gac tgc tgccac aac cag tgt gct gca ggc tgc 720 Gly Lys Ser Pro Ser Asp Cys Cys HisAsn Gln Cys Ala Ala Gly Cys 225 230 235 240 aca ggc ccc cgg gag agc gactgc ctg gtc tgc cgc aaa ttc cga gac 768 Thr Gly Pro Arg Glu Ser Asp CysLeu Val Cys Arg Lys Phe Arg Asp 245 250 255 gaa gcc acg tgc aag gac acctgc ccc cca ctc atg ctc tac aac ccc 816 Glu Ala Thr Cys Lys Asp Thr CysPro Pro Leu Met Leu Tyr Asn Pro 260 265 270 acc acg tac cag atg gat gtgaac ccc gag ggc aaa tac agc ttt ggt 864 Thr Thr Tyr Gln Met Asp Val AsnPro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 gcc acc tgc gtg aag aag tgtccc cgt aat tat gtg gtg aca gat cac 912 Ala Thr Cys Val Lys Lys Cys ProArg Asn Tyr Val Val Thr Asp His 290 295 300 ggc tcg tgc gtc cga gcc tgtggg gcc gac agc tat gag atg gag gaa 960 Gly Ser Cys Val Arg Ala Cys GlyAla Asp Ser Tyr Glu Met Glu Glu 305 310 315 320 gac ggc gtc cgc aag tgtaag aag tgc gaa ggg cct tgc cgc aaa gtg 1008 Asp Gly Val Arg Lys Cys LysLys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 tgt aac gga ata ggt attggt gaa ttt aaa gac tca ctc tcc ata aat 1056 Cys Asn Gly Ile Gly Ile GlyGlu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 gct acg aat att aaa cacttc aaa aac tgc acc tcc atc agt ggc gat 1104 Ala Thr Asn Ile Lys His PheLys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 ctc cac atc ctg ccg gtggca ttt agg ggt gac tcc ttc aca cat act 1152 Leu His Ile Leu Pro Val AlaPhe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 cct cct ctg gat cca caggaa ctg gat att ctg aaa acc gta aag gaa 1200 Pro Pro Leu Asp Pro Gln GluLeu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 atc aca ggg ttt ttgctg att cag gct tgg cct gaa aac agg acg gac 1248 Ile Thr Gly Phe Leu LeuIle Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 ctc cat gcc ttt gagaac cta gaa atc ata cgc ggc agg acc aag caa 1296 Leu His Ala Phe Glu AsnLeu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 cat ggt cag ttt tctctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344 His Gly Gln Phe Ser LeuAla Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445 gga tta cgc tcc ctcaag gag ata agt gat gga gat gtg ata att tca 1392 Gly Leu Arg Ser Leu LysGlu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 gga aac aaa aat ttgtgc tat gca aat aca ata aac tgg aaa aaa ctg 1440 Gly Asn Lys Asn Leu CysTyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 ttt ggg acc tccggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488 Phe Gly Thr Ser GlyGln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 aac agc tgc aaggcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536 Asn Ser Cys Lys AlaThr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510 gag ggc tgc tggggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584 Glu Gly Cys Trp GlyPro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525 gtc agc cga ggcagg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632 Val Ser Arg Gly ArgGlu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540 gag cca agg gagttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680 Glu Pro Arg Glu PheVal Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 gag tgc ctgcct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728 Glu Cys Leu ProGln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 gac aac tgtatc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776 Asp Asn Cys IleGln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 aag acc tgcccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824 Lys Thr Cys ProAla Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 aag tac gcagac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872 Lys Tyr Ala AspAla Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 acc tac ggatgc act ggg cca ggt ctt gaa ggc tgt cca acg aat ggg 1920 Thr Tyr Gly CysThr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635 640 cct aagatc ccg tcc atc gcc act ggg atg gtg ggg gcc ctc ctc ttg 1968 Pro Lys IlePro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 645 650 655 ctg ctggtg gtg gcc ctg ggg atc ggc ctc ttc atg cga agg cgc cac 2016 Leu Leu ValVal Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His 660 665 670 atc gttcgg aag cgc acg ctg cgg agg ctg ctg cag gag agg gag ctt 2064 Ile Val ArgLys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675 680 685 gtg gagcct ctt aca ccc agt gga gaa gct ccc aac caa gct ctc ttg 2112 Val Glu ProLeu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu 690 695 700 agg atcttg aag gaa act gaa ttc aaa aag atc aaa gtg ctg ggc tcc 2160 Arg Ile LeuLys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser 705 710 715 720 ggtgcg ttc ggc acg gtg tat aag gga ctc tgg atc cca gaa ggt gag 2208 Gly AlaPhe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu 725 730 735 aaagtt aaa att ccc gtc gct atc aag gaa tta aga gaa gca aca tct 2256 Lys ValLys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750 ccgaaa gcc aac aag gaa atc ctc gat gaa gcc tac gtg atg gcc agc 2304 Pro LysAla Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760 765 gtggac aac ccc cac gtg tgc cgc ctg ctg ggc atc tgc ctc acc tcc 2352 Val AspAsn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780 accgtg cag ctc atc acg cag ctc atg ccc ttc ggc tgc ctc ctg gac 2400 Thr ValGln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp 785 790 795 800tat gtc cgg gaa cac aaa gac aat att ggc tcc cag tac ctg ctc aac 2448 TyrVal Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805 810 815tgg tgt gtg cag atc gca aag ggc atg aac tac ttg gag gac cgt cgc 2496 TrpCys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg 820 825 830ttg gtg cac cgc gac ctg gca gcc agg aac gta ctg gtg aaa aca ccg 2544 LeuVal His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro 835 840 845cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa ctg ctg ggt gcg 2592 GlnHis Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala 850 855 860gaa gag aaa gaa tac cat gca gaa gga ggc aaa gtg cct atc aag tgg 2640 GluGlu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp 865 870 875880 atg gca ttg gaa tca att tta cac aga atc tat acc cac cag agt gat 2688Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885 890895 gtc tgg agc tac ggg gtg acc gtt tgg gag ttg atg acc ttt gga tcc 2736Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905910 aag cca tat gac gga atc cct gcc agc gag atc tcc tcc atc ctg gag 2784Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920925 aaa gga gaa cgc ctc cct cag cca ccc ata tgt acc atc gat gtc tac 2832Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr 930 935940 atg atc atg gtc aag tgc tgg atg ata gac gca gat agt cgc cca aag 2880Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys 945 950955 960 ttc cgt gag ttg atc atc gaa ttc tcc aaa atg gcc cga gac ccc cag2928 Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln 965970 975 cgc tac ctt gtc att cag ggg gat gaa aga atg cat ttg cca agt cct2976 Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro 980985 990 aca gac tcc aac ttc tac cgt gcc ctg atg gat gaa gaa gac atg gac3024 Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp 9951000 1005 gac gtg gtg gat gcc gac gag tac ctc atc cca cag cag ggc ttc3069 Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe 10101015 1020 ttc agc agc ccc tcc acg tca cgg act ccc ctc ctg agc tct ctg3114 Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu 10251030 1035 agt gca acc agc aac aat tcc acc gtg gct tgc att gat aga aat3159 Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn 10401045 1050 ggg ctg caa agc tgt ccc atc aag gaa gac agc ttc ttg cag cga3204 Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg 10551060 1065 tac agc tca gac ccc aca ggc gcc ttg act gag gac agc ata gac3249 Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp 10701075 1080 gac acc ttc ctc cca gtg cct gaa tac ata aac cag tcc gtt ccc3294 Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro 10851090 1095 aaa agg ccc gct ggc tct gtg cag aat cct gtc tat cac aat cag3339 Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln 11001105 1110 cct ctg aac ccc gcg ccc agc aga gac cca cac tac cag gac ccc3384 Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro 11151120 1125 cac agc act gca gtg ggc aac ccc gag tat ctc aac act gtc cag3429 His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln 11301135 1140 ccc acc tgt gtc aac agc aca ttc gac agc cct gcc cac tgg gcc3474 Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala 11451150 1155 cag aaa ggc agc cac caa att agc ctg gac aac cct gac tac cag3519 Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln 11601165 1170 cag gac ttc ttt ccc aag gaa gcc aag cca aat ggc atc ttt aag3564 Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys 11751180 1185 ggc tcc aca gct gaa aat gca gaa tac cta agg gtc gcg cca caa3609 Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln 11901195 1200 agc agt gaa ttt att gga gca tga 3633 Ser Ser Glu Phe Ile GlyAla 1205 1210 2 1210 PRT Human(homo sapiens) 2 Met Arg Pro Ser Gly ThrAla Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro AlaSer Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn LysLeu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln ArgMet Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr TyrVal Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln GluVal Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile ProLeu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu AsnSer Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys ThrGly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 HisGly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155160 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165170 175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn CysGln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly ArgCys Arg 210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys AlaAla Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val CysArg Lys Phe Arg Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro ProLeu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn ProGlu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys ProArg Asn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala CysGly Ala Asp Ser Tyr Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg LysCys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly IleGly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr AsnIle Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu HisIle Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 ProPro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405410 415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr SerLeu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val IleIle Ser 450 455 460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn TrpLys Lys Leu 465 470 475 480 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile IleSer Asn Arg Gly Glu 485 490 495 Asn Ser Cys Lys Ala Thr Gly Gln Val CysHis Ala Leu Cys Ser Pro 500 505 510 Glu Gly Cys Trp Gly Pro Glu Pro ArgAsp Cys Val Ser Cys Arg Asn 515 520 525 Val Ser Arg Gly Arg Glu Cys ValAsp Lys Cys Asn Leu Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe Val GluAsn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys Leu Pro GlnAla Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys IleGln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys Thr CysPro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys TyrAla Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 ThrTyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635640 Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 645650 655 Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His660 665 670 Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg GluLeu 675 680 685 Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln AlaLeu Leu 690 695 700 Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys ValLeu Gly Ser 705 710 715 720 Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu TrpIle Pro Glu Gly Glu 725 730 735 Lys Val Lys Ile Pro Val Ala Ile Lys GluLeu Arg Glu Ala Thr Ser 740 745 750 Pro Lys Ala Asn Lys Glu Ile Leu AspGlu Ala Tyr Val Met Ala Ser 755 760 765 Val Asp Asn Pro His Val Cys ArgLeu Leu Gly Ile Cys Leu Thr Ser 770 775 780 Thr Val Gln Leu Ile Thr GlnLeu Met Pro Phe Gly Cys Leu Leu Asp 785 790 795 800 Tyr Val Arg Glu HisLys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805 810 815 Trp Cys Val GlnIle Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg 820 825 830 Leu Val HisArg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro 835 840 845 Gln HisVal Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala 850 855 860 GluGlu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp 865 870 875880 Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885890 895 Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser900 905 910 Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile LeuGlu 915 920 925 Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile AspVal Tyr 930 935 940 Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp SerArg Pro Lys 945 950 955 960 Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys MetAla Arg Asp Pro Gln 965 970 975 Arg Tyr Leu Val Ile Gln Gly Asp Glu ArgMet His Leu Pro Ser Pro 980 985 990 Thr Asp Ser Asn Phe Tyr Arg Ala LeuMet Asp Glu Glu Asp Met Asp 995 1000 1005 Asp Val Val Asp Ala Asp GluTyr Leu Ile Pro Gln Gln Gly Phe 1010 1015 1020 Phe Ser Ser Pro Ser ThrSer Arg Thr Pro Leu Leu Ser Ser Leu 1025 1030 1035 Ser Ala Thr Ser AsnAsn Ser Thr Val Ala Cys Ile Asp Arg Asn 1040 1045 1050 Gly Leu Gln SerCys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg 1055 1060 1065 Tyr Ser SerAsp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp 1070 1075 1080 Asp ThrPhe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro 1085 1090 1095 LysArg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln 1100 1105 1110Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro 1115 11201125 His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln 11301135 1140 Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala1145 1150 1155 Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp TyrGln 1160 1165 1170 Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly IlePhe Lys 1175 1180 1185 Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg ValAla Pro Gln 1190 1195 1200 Ser Ser Glu Phe Ile Gly Ala 1205 1210 3 1974DNA Human(homo sapiens) CDS (1)..(1971) 3 atg cga ccc tcc ggg acg gccggg gca gcg ctc ctg gcg ctg ctg gct 48 Met Arg Pro Ser Gly Thr Ala GlyAla Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 gcg ctc tgc ccg gcg agt cgggct ctg gag gaa aag aaa gtt tgc caa 96 Ala Leu Cys Pro Ala Ser Arg AlaLeu Glu Glu Lys Lys Val Cys Gln 20 25 30 ggc acg agt aac aag ctc acg cagttg ggc act ttt gaa gat cat ttt 144 Gly Thr Ser Asn Lys Leu Thr Gln LeuGly Thr Phe Glu Asp His Phe 35 40 45 ctc agc ctc cag agg atg ttc aat aactgt gag gtg gtc ctt ggg aat 192 Leu Ser Leu Gln Arg Met Phe Asn Asn CysGlu Val Val Leu Gly Asn 50 55 60 ttg gaa att acc tat gtg cag agg aat tatgat ctt tcc ttc tta aag 240 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr AspLeu Ser Phe Leu Lys 65 70 75 80 acc atc cag gag gtg gct ggt tat gtc ctcatt gcc ctc aac aca gtg 288 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu IleAla Leu Asn Thr Val 85 90 95 gag cga att cct ttg gaa aac ctg cag atc atcaga gga aat atg tac 336 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile ArgGly Asn Met Tyr 100 105 110 tac gaa aat tcc tat gcc tta gca gtc tta tctaac tat gat gca aat 384 Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser AsnTyr Asp Ala Asn 115 120 125 aaa acc gga ctg aag gag ctg ccc atg aga aattta cag gaa atc ctg 432 Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn LeuGln Glu Ile Leu 130 135 140 cat ggc gcc gtg cgg ttc agc aac aac cct gccctg tgc aac gtg gag 480 His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala LeuCys Asn Val Glu 145 150 155 160 agc atc cag tgg cgg gac ata gtc agc agtgac ttt ctc agc aac atg 528 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser AspPhe Leu Ser Asn Met 165 170 175 tcg atg gac ttc cag aac cac ctg ggc agctgc caa aag tgt gat cca 576 Ser Met Asp Phe Gln Asn His Leu Gly Ser CysGln Lys Cys Asp Pro 180 185 190 agc tgt ccc aat ggg agc tgc tgg ggt gcagga gag gag aac tgc cag 624 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala GlyGlu Glu Asn Cys Gln 195 200 205 aaa ctg acc aaa atc atc tgt gcc cag cagtgc tcc ggg cgc tgc cgt 672 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln CysSer Gly Arg Cys Arg 210 215 220 ggc aag tcc ccc agt gac tgc tgc cac aaccag tgt gct gca ggc tgc 720 Gly Lys Ser Pro Ser Asp Cys Cys His Asn GlnCys Ala Ala Gly Cys 225 230 235 240 aca ggc ccc cgg gag agc gac tgc ctggtc tgc cgc aaa ttc cga gac 768 Thr Gly Pro Arg Glu Ser Asp Cys Leu ValCys Arg Lys Phe Arg Asp 245 250 255 gaa gcc acg tgc aag gac acc tgc ccccca ctc atg ctc tac aac ccc 816 Glu Ala Thr Cys Lys Asp Thr Cys Pro ProLeu Met Leu Tyr Asn Pro 260 265 270 acc acg tac cag atg gat gtg aac cccgag ggc aaa tac agc ttt ggt 864 Thr Thr Tyr Gln Met Asp Val Asn Pro GluGly Lys Tyr Ser Phe Gly 275 280 285 gcc acc tgc gtg aag aag tgt ccc cgtaat tat gtg gtg aca gat cac 912 Ala Thr Cys Val Lys Lys Cys Pro Arg AsnTyr Val Val Thr Asp His 290 295 300 ggc tcg tgc gtc cga gcc tgt ggg gccgac agc tat gag atg gag gaa 960 Gly Ser Cys Val Arg Ala Cys Gly Ala AspSer Tyr Glu Met Glu Glu 305 310 315 320 gac ggc gtc cgc aag tgt aag aagtgc gaa ggg cct tgc cgc aaa gtg 1008 Asp Gly Val Arg Lys Cys Lys Lys CysGlu Gly Pro Cys Arg Lys Val 325 330 335 tgt aac gga ata ggt att ggt gaattt aaa gac tca ctc tcc ata aat 1056 Cys Asn Gly Ile Gly Ile Gly Glu PheLys Asp Ser Leu Ser Ile Asn 340 345 350 gct acg aat att aaa cac ttc aaaaac tgc acc tcc atc agt ggc gat 1104 Ala Thr Asn Ile Lys His Phe Lys AsnCys Thr Ser Ile Ser Gly Asp 355 360 365 ctc cac atc ctg ccg gtg gca tttagg ggt gac tcc ttc aca cat act 1152 Leu His Ile Leu Pro Val Ala Phe ArgGly Asp Ser Phe Thr His Thr 370 375 380 cct cct ctg gat cca cag gaa ctggat att ctg aaa acc gta aag gaa 1200 Pro Pro Leu Asp Pro Gln Glu Leu AspIle Leu Lys Thr Val Lys Glu 385 390 395 400 atc aca ggg ttt ttg ctg attcag gct tgg cct gaa aac agg acg gac 1248 Ile Thr Gly Phe Leu Leu Ile GlnAla Trp Pro Glu Asn Arg Thr Asp 405 410 415 ctc cat gcc ttt gag aac ctagaa atc ata cgc ggc agg acc aag caa 1296 Leu His Ala Phe Glu Asn Leu GluIle Ile Arg Gly Arg Thr Lys Gln 420 425 430 cat ggt cag ttt tct ctt gcagtc gtc agc ctg aac ata aca tcc ttg 1344 His Gly Gln Phe Ser Leu Ala ValVal Ser Leu Asn Ile Thr Ser Leu 435 440 445 gga tta cgc tcc ctc aag gagata agt gat gga gat gtg ata att tca 1392 Gly Leu Arg Ser Leu Lys Glu IleSer Asp Gly Asp Val Ile Ile Ser 450 455 460 gga aac aaa aat ttg tgc tatgca aat aca ata aac tgg aaa aaa ctg 1440 Gly Asn Lys Asn Leu Cys Tyr AlaAsn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 ttt ggg acc tcc ggt cagaaa acc aaa att ata agc aac aga ggt gaa 1488 Phe Gly Thr Ser Gly Gln LysThr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 aac agc tgc aag gcc acaggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536 Asn Ser Cys Lys Ala Thr GlyGln Val Cys His Ala Leu Cys Ser Pro 500 505 510 gag ggc tgc tgg ggc ccggag ccc agg gac tgc gtc tct tgc cgg aat 1584 Glu Gly Cys Trp Gly Pro GluPro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525 gtc agc cga ggc agg gaatgc gtg gac aag tgc aac ctt ctg gag ggt 1632 Val Ser Arg Gly Arg Glu CysVal Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540 gag cca agg gag ttt gtggag aac tct gag tgc ata cag tgc cac cca 1680 Glu Pro Arg Glu Phe Val GluAsn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 gag tgc ctg cct caggcc atg aac atc acc tgc aca gga cgg gga cca 1728 Glu Cys Leu Pro Gln AlaMet Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 gac aac tgt atc cagtgt gcc cac tac att gac ggc ccc cac tgc gtc 1776 Asp Asn Cys Ile Gln CysAla His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 aag acc tgc ccg gcagga gtc atg gga gaa aac aac acc ctg gtc tgg 1824 Lys Thr Cys Pro Ala GlyVal Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 aag tac gca gac gccggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872 Lys Tyr Ala Asp Ala GlyHis Val Cys His Leu Cys His Pro Asn Cys 610 615 620 acc tac gga tgc actggg cca ggt ctt gaa ggc tgt cca acg aat gga 1920 Thr Tyr Gly Cys Thr GlyPro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635 640 agc tac ata gtgtct cac ttt cca aga tca ttc tac aag atg tca gtg 1968 Ser Tyr Ile Val SerHis Phe Pro Arg Ser Phe Tyr Lys Met Ser Val 645 650 655 cac tga 1974 His4 657 PRT Human homo sapiens) 4 Met Arg Pro Ser Gly Thr Ala Gly Ala AlaLeu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro Ala Ser Arg Ala LeuGlu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn Lys Leu Thr Gln LeuGly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln Arg Met Phe Asn AsnCys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr Tyr Val Gln Arg AsnTyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu Val Ala Gly TyrVal Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu Asn LeuGln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu Asn Ser Tyr Ala LeuAla Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys Thr Gly Leu Lys GluLeu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 His Gly Ala Val ArgPhe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155 160 Ser Ile GlnTrp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175 Ser MetAsp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190 SerCys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe ArgAsp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu TyrAsn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys TyrSer Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr ValVal Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp SerTyr Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys CysGlu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly GluPhe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr Asn Ile Lys His PheLys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile Leu Pro ValAla Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro Pro Leu Asp ProGln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 Ile Thr GlyPhe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 Leu HisAla Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 HisGly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465470 475 480 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg GlyGlu 485 490 495 Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu CysSer Pro 500 505 510 Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val SerCys Arg Asn 515 520 525 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys AsnLeu Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu CysIle Gln Cys His Pro 545 550 555 560 Glu Cys Leu Pro Gln Ala Met Asn IleThr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala HisTyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly ValMet Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala Asp Ala GlyHis Val Cys His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr Gly Cys ThrGly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635 640 Ser Tyr IleVal Ser His Phe Pro Arg Ser Phe Tyr Lys Met Ser Val 645 650 655 His 52118 DNA Human(homo sapiens) CDS (1)..(2115) 5 atg cga ccc tcc ggg acggcc ggg gca gcg ctc ctg gcg ctg ctg gct 48 Met Arg Pro Ser Gly Thr AlaGly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 gcg ctc tgc ccg gcg agtcgg gct ctg gag gaa aag aaa gtt tgc caa 96 Ala Leu Cys Pro Ala Ser ArgAla Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 ggc acg agt aac aag ctc acgcag ttg ggc act ttt gaa gat cat ttt 144 Gly Thr Ser Asn Lys Leu Thr GlnLeu Gly Thr Phe Glu Asp His Phe 35 40 45 ctc agc ctc cag agg atg ttc aataac tgt gag gtg gtc ctt ggg aat 192 Leu Ser Leu Gln Arg Met Phe Asn AsnCys Glu Val Val Leu Gly Asn 50 55 60 ttg gaa att acc tat gtg cag agg aattat gat ctt tcc ttc tta aag 240 Leu Glu Ile Thr Tyr Val Gln Arg Asn TyrAsp Leu Ser Phe Leu Lys 65 70 75 80 acc atc cag gag gtg gct ggt tat gtcctc att gcc ctc aac aca gtg 288 Thr Ile Gln Glu Val Ala Gly Tyr Val LeuIle Ala Leu Asn Thr Val 85 90 95 gag cga att cct ttg gaa aac ctg cag atcatc aga gga aat atg tac 336 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile IleArg Gly Asn Met Tyr 100 105 110 tac gaa aat tcc tat gcc tta gca gtc ttatct aac tat gat gca aat 384 Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu SerAsn Tyr Asp Ala Asn 115 120 125 aaa acc gga ctg aag gag ctg ccc atg agaaat tta cag gaa atc ctg 432 Lys Thr Gly Leu Lys Glu Leu Pro Met Arg AsnLeu Gln Glu Ile Leu 130 135 140 cat ggc gcc gtg cgg ttc agc aac aac cctgcc ctg tgc aac gtg gag 480 His Gly Ala Val Arg Phe Ser Asn Asn Pro AlaLeu Cys Asn Val Glu 145 150 155 160 agc atc cag tgg cgg gac ata gtc agcagt gac ttt ctc agc aac atg 528 Ser Ile Gln Trp Arg Asp Ile Val Ser SerAsp Phe Leu Ser Asn Met 165 170 175 tcg atg gac ttc cag aac cac ctg ggcagc tgc caa aag tgt gat cca 576 Ser Met Asp Phe Gln Asn His Leu Gly SerCys Gln Lys Cys Asp Pro 180 185 190 agc tgt ccc aat ggg agc tgc tgg ggtgca gga gag gag aac tgc cag 624 Ser Cys Pro Asn Gly Ser Cys Trp Gly AlaGly Glu Glu Asn Cys Gln 195 200 205 aaa ctg acc aaa atc atc tgt gcc cagcag tgc tcc ggg cgc tgc cgt 672 Lys Leu Thr Lys Ile Ile Cys Ala Gln GlnCys Ser Gly Arg Cys Arg 210 215 220 ggc aag tcc ccc agt gac tgc tgc cacaac cag tgt gct gca ggc tgc 720 Gly Lys Ser Pro Ser Asp Cys Cys His AsnGln Cys Ala Ala Gly Cys 225 230 235 240 aca ggc ccc cgg gag agc gac tgcctg gtc tgc cgc aaa ttc cga gac 768 Thr Gly Pro Arg Glu Ser Asp Cys LeuVal Cys Arg Lys Phe Arg Asp 245 250 255 gaa gcc acg tgc aag gac acc tgcccc cca ctc atg ctc tac aac ccc 816 Glu Ala Thr Cys Lys Asp Thr Cys ProPro Leu Met Leu Tyr Asn Pro 260 265 270 acc acg tac cag atg gat gtg aacccc gag ggc aaa tac agc ttt ggt 864 Thr Thr Tyr Gln Met Asp Val Asn ProGlu Gly Lys Tyr Ser Phe Gly 275 280 285 gcc acc tgc gtg aag aag tgt ccccgt aat tat gtg gtg aca gat cac 912 Ala Thr Cys Val Lys Lys Cys Pro ArgAsn Tyr Val Val Thr Asp His 290 295 300 ggc tcg tgc gtc cga gcc tgt ggggcc gac agc tat gag atg gag gaa 960 Gly Ser Cys Val Arg Ala Cys Gly AlaAsp Ser Tyr Glu Met Glu Glu 305 310 315 320 gac ggc gtc cgc aag tgt aagaag tgc gaa ggg cct tgc cgc aaa gtg 1008 Asp Gly Val Arg Lys Cys Lys LysCys Glu Gly Pro Cys Arg Lys Val 325 330 335 tgt aac gga ata ggt att ggtgaa ttt aaa gac tca ctc tcc ata aat 1056 Cys Asn Gly Ile Gly Ile Gly GluPhe Lys Asp Ser Leu Ser Ile Asn 340 345 350 gct acg aat att aaa cac ttcaaa aac tgc acc tcc atc agt ggc gat 1104 Ala Thr Asn Ile Lys His Phe LysAsn Cys Thr Ser Ile Ser Gly Asp 355 360 365 ctc cac atc ctg ccg gtg gcattt agg ggt gac tcc ttc aca cat act 1152 Leu His Ile Leu Pro Val Ala PheArg Gly Asp Ser Phe Thr His Thr 370 375 380 cct cct ctg gat cca cag gaactg gat att ctg aaa acc gta aag gaa 1200 Pro Pro Leu Asp Pro Gln Glu LeuAsp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 atc aca ggg ttt ttg ctgatt cag gct tgg cct gaa aac agg acg gac 1248 Ile Thr Gly Phe Leu Leu IleGln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 ctc cat gcc ttt gag aaccta gaa atc ata cgc ggc agg acc aag caa 1296 Leu His Ala Phe Glu Asn LeuGlu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 cat ggt cag ttt tct cttgca gtc gtc agc ctg aac ata aca tcc ttg 1344 His Gly Gln Phe Ser Leu AlaVal Val Ser Leu Asn Ile Thr Ser Leu 435 440 445 gga tta cgc tcc ctc aaggag ata agt gat gga gat gtg ata att tca 1392 Gly Leu Arg Ser Leu Lys GluIle Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 gga aac aaa aat ttg tgctat gca aat aca ata aac tgg aaa aaa ctg 1440 Gly Asn Lys Asn Leu Cys TyrAla Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 ttt ggg acc tcc ggtcag aaa acc aaa att ata agc aac aga ggt gaa 1488 Phe Gly Thr Ser Gly GlnLys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 aac agc tgc aag gccaca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536 Asn Ser Cys Lys Ala ThrGly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510 gag ggc tgc tgg ggcccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584 Glu Gly Cys Trp Gly ProGlu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525 gtc agc cga ggc agggaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632 Val Ser Arg Gly Arg GluCys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540 gag cca agg gag tttgtg gag aac tct gag tgc ata cag tgc cac cca 1680 Glu Pro Arg Glu Phe ValGlu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 gag tgc ctg cctcag gcc atg aac atc acc tgc aca gga cgg gga cca 1728 Glu Cys Leu Pro GlnAla Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 gac aac tgt atccag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776 Asp Asn Cys Ile GlnCys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 aag acc tgc ccggca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824 Lys Thr Cys Pro AlaGly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 aag tac gca gacgcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872 Lys Tyr Ala Asp AlaGly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 acc tac ggg ccagga aat gag agt ctc aaa gcc atg tta ttc tgc ctt 1920 Thr Tyr Gly Pro GlyAsn Glu Ser Leu Lys Ala Met Leu Phe Cys Leu 625 630 635 640 ttt aaa ctatca tcc tgt aat caa agt aat gat ggc agc gtg tcc cac 1968 Phe Lys Leu SerSer Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His 645 650 655 cag agc gggagc cca gct gct cag gag tca tgc tta gga tgg atc cct 2016 Gln Ser Gly SerPro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro 660 665 670 tct ctt ctgccg tca gag ttt cag ctg ggt tgg ggt gga tgc agc cac 2064 Ser Leu Leu ProSer Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His 675 680 685 ctc cat gcctgg cct tct gca tct gtg atc atc acg gcc tcc tcc tgc 2112 Leu His Ala TrpPro Ser Ala Ser Val Ile Ile Thr Ala Ser Ser Cys 690 695 700 cac tga 2118His 705 6 705 PRT Human(homo sapiens) 6 Met Arg Pro Ser Gly Thr Ala GlyAla Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro Ala Ser ArgAla Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn Lys Leu ThrGln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln Arg Met PheAsn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr Tyr Val GlnArg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu Val AlaGly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu GluAsn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu Asn Ser TyrAla Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys Thr Gly LeuLys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 His Gly AlaVal Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155 160 SerIle Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala GlyCys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg LysPhe Arg Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu MetLeu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu GlyLys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys Pro Arg AsnTyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala Cys Gly AlaAsp Ser Tyr Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg Lys Cys LysLys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile Gly IleGly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr Asn Ile LysHis Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile LeuPro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro Pro LeuAsp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 IleThr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450 455 460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys LysLeu 465 470 475 480 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser AsnArg Gly Glu 485 490 495 Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His AlaLeu Cys Ser Pro 500 505 510 Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp CysVal Ser Cys Arg Asn 515 520 525 Val Ser Arg Gly Arg Glu Cys Val Asp LysCys Asn Leu Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe Val Glu Asn SerGlu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys Leu Pro Gln Ala MetAsn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys Ile Gln CysAla His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys Thr Cys Pro AlaGly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala AspAla Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr GlyPro Gly Asn Glu Ser Leu Lys Ala Met Leu Phe Cys Leu 625 630 635 640 PheLys Leu Ser Ser Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His 645 650 655Gln Ser Gly Ser Pro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro 660 665670 Ser Leu Leu Pro Ser Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His 675680 685 Leu His Ala Trp Pro Ser Ala Ser Val Ile Ile Thr Ala Ser Ser Cys690 695 700 His 705 7 1887 DNA Human homo sapiens) CDS (1)..(1884) 7 atgcga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48 Met ArgPro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 gcgctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc caa 96 Ala LeuCys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 ggc acgagt aac aag ctc acg cag ttg ggc act ttt gaa gat cat ttt 144 Gly Thr SerAsn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 ctc agc ctccag agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat 192 Leu Ser Leu GlnArg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 ttg gaa att acctat gtg cag agg aat tat gat ctt tcc ttc tta aag 240 Leu Glu Ile Thr TyrVal Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 acc atc cag gaggtg gct ggt tat gtc ctc att gcc ctc aac aca gtg 288 Thr Ile Gln Glu ValAla Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 gag cga att cct ttggaa aac ctg cag atc atc aga gga aat atg tac 336 Glu Arg Ile Pro Leu GluAsn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 tac gaa aat tcc tatgcc tta gca gtc tta tct aac tat gat gca aat 384 Tyr Glu Asn Ser Tyr AlaLeu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 aaa acc gga ctg aaggag ctg ccc atg aga aat tta cag gaa atc ctg 432 Lys Thr Gly Leu Lys GluLeu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 cat ggc gcc gtg cggttc agc aac aac cct gcc ctg tgc aac gtg gag 480 His Gly Ala Val Arg PheSer Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155 160 agc atc cag tggcgg gac ata gtc agc agt gac ttt ctc agc aac atg 528 Ser Ile Gln Trp ArgAsp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175 tcg atg gac ttccag aac cac ctg ggc agc tgc caa aag tgt gat cca 576 Ser Met Asp Phe GlnAsn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190 agc tgt ccc aatggg agc tgc tgg ggt gca gga gag gag aac tgc cag 624 Ser Cys Pro Asn GlySer Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205 aaa ctg acc aaaatc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt 672 Lys Leu Thr Lys IleIle Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220 ggc aag tcc cccagt gac tgc tgc cac aac cag tgt gct gca ggc tgc 720 Gly Lys Ser Pro SerAsp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 aca ggc ccccgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac 768 Thr Gly Pro ArgGlu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 gaa gcc acgtgc aag gac acc tgc ccc cca ctc atg ctc tac aac ccc 816 Glu Ala Thr CysLys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 acc acg taccag atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt 864 Thr Thr Tyr GlnMet Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 gcc acc tgcgtg aag aag tgt ccc cgt aat tat gtg gtg aca gat cac 912 Ala Thr Cys ValLys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300 ggc tcg tgcgtc cga gcc tgt ggg gcc gac agc tat gag atg gag gaa 960 Gly Ser Cys ValArg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu 305 310 315 320 gac ggcgtc cgc aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg 1008 Asp Gly ValArg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 tgt aacgga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat 1056 Cys Asn GlyIle Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 gct acgaat att aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat 1104 Ala Thr AsnIle Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 ctc cacatc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat act 1152 Leu His IleLeu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 cct cctctg gat cca cag gaa ctg gat att ctg aaa acc gta aag gaa 1200 Pro Pro LeuAsp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 atcaca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg gac 1248 Ile ThrGly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 ctccat gcc ttt gag aac cta gaa atc ata cgc ggc agg acc aag caa 1296 Leu HisAla Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 catggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg 1344 His GlyGln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445 ggatta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att tca 1392 Gly LeuArg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 ggaaac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg 1440 Gly AsnLys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt gaa 1488 PheGly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc 1536 AsnSer Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat 1584 GluGly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt 1632 ValSer Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac cca 1680 GluPro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555560 gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570575 gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585590 aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600605 aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc 1872Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615620 acc tac ggg tcc taa 1887 Thr Tyr Gly Ser 625 8 628 PRT Human(homosapiens) 8 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu LeuAla 1 5 10 15 Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys ValCys Gln 20 25 30 Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu AspHis Phe 35 40 45 Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val LeuGly Asn 50 55 60 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser PheLeu Lys 65 70 75 80 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala LeuAsn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg GlyAsn Met Tyr 100 105 110 Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser AsnTyr Asp Ala Asn 115 120 125 Lys Thr Gly Leu Lys Glu Leu Pro Met Arg AsnLeu Gln Glu Ile Leu 130 135 140 His Gly Ala Val Arg Phe Ser Asn Asn ProAla Leu Cys Asn Val Glu 145 150 155 160 Ser Ile Gln Trp Arg Asp Ile ValSer Ser Asp Phe Leu Ser Asn Met 165 170 175 Ser Met Asp Phe Gln Asn HisLeu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190 Ser Cys Pro Asn Gly SerCys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205 Lys Leu Thr Lys IleIle Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220 Gly Lys Ser ProSer Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 Thr GlyPro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 GluAla Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290295 300 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys ArgLys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser LeuSer Ile Asn 340 345 350 Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr SerIle Ser Gly Asp 355 360 365 Leu His Ile Leu Pro Val Ala Phe Arg Gly AspSer Phe Thr His Thr 370 375 380 Pro Pro Leu Asp Pro Gln Glu Leu Asp IleLeu Lys Thr Val Lys Glu 385 390 395 400 Ile Thr Gly Phe Leu Leu Ile GlnAla Trp Pro Glu Asn Arg Thr Asp 405 410 415 Leu His Ala Phe Glu Asn LeuGlu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 His Gly Gln Phe Ser LeuAla Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445 Gly Leu Arg Ser LeuLys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 Gly Asn Lys AsnLeu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 Phe GlyThr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 AsnSer Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520525 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530535 540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly ArgGly Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly ProHis Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn AsnThr Leu Val Trp 595 600 605 Lys Tyr Ala Asp Ala Gly His Val Cys His LeuCys His Pro Asn Cys 610 615 620 Thr Tyr Gly Ser 625 9 1218 DNA Humanhomo sapiens) CDS (1)..(1215) 9 atg cga ccc tcc ggg acg gcc ggg gca gcgctc ctg gcg ctg ctg gct 48 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala LeuLeu Ala Leu Leu Ala 1 5 10 15 gcg ctc tgc ccg gcg agt cgg gct ctg gaggaa aag aaa gtt tgc caa 96 Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu GluLys Lys Val Cys Gln 20 25 30 ggc acg agt aac aag ctc acg cag ttg ggc actttt gaa gat cat ttt 144 Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr PheGlu Asp His Phe 35 40 45 ctc agc ctc cag agg atg ttc aat aac tgt gag gtggtc ctt ggg aat 192 Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val ValLeu Gly Asn 50 55 60 ttg gaa att acc tat gtg cag agg aat tat gat ctt tccttc tta aag 240 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser PheLeu Lys 65 70 75 80 acc atc cag gag gtg gct ggt tat gtc ctc att gcc ctcaac aca gtg 288 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu AsnThr Val 85 90 95 gag cga att cct ttg gaa aac ctg cag atc atc aga gga aatatg tac 336 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn MetTyr 100 105 110 tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gatgca aat 384 Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp AlaAsn 115 120 125 aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaaatc ctg 432 Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu IleLeu 130 135 140 cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aacgtg gag 480 His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn ValGlu 145 150 155 160 agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctcagc aac atg 528 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu SerAsn Met 165 170 175 tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aagtgt gat cca 576 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys CysAsp Pro 180 185 190 agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gagaac tgc cag 624 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu AsnCys Gln 195 200 205 aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc gggcgc tgc cgt 672 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly ArgCys Arg 210 215 220 ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gctgca ggc tgc 720 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala AlaGly Cys 225 230 235 240 aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgcaaa ttc cga gac 768 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg LysPhe Arg Asp 245 250 255 gaa gcc acg tgc aag gac acc tgc ccc cca ctc atgctc tac aac ccc 816 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met LeuTyr Asn Pro 260 265 270 acc acg tac cag atg gat gtg aac ccc gag ggc aaatac agc ttt ggt 864 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys TyrSer Phe Gly 275 280 285 gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtggtg aca gat cac 912 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val ValThr Asp His 290 295 300 ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tatgag atg gag gaa 960 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr GluMet Glu Glu 305 310 315 320 gac ggc gtc cgc aag tgt aag aag tgc gaa gggcct tgc cgc aaa gtg 1008 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly ProCys Arg Lys Val 325 330 335 tgt aac gga ata ggt att ggt gaa ttt aaa gactca ctc tcc ata aat 1056 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp SerLeu Ser Ile Asn 340 345 350 gct acg aat att aaa cac ttc aaa aac tgc acctcc atc agt ggc gat 1104 Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr SerIle Ser Gly Asp 355 360 365 ctc cac atc ctg ccg gtg gca ttt agg ggt gactcc ttc aca cat act 1152 Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp SerPhe Thr His Thr 370 375 380 cct cct ctg gat cca cag gaa ctg gat att ctgaaa acc gta aag gaa 1200 Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu LysThr Val Lys Glu 385 390 395 400 atc aca ggt ttg agc tga 1218 Ile Thr GlyLeu Ser 405 10 405 PRT Human (homo sapiens) 10 Met Arg Pro Ser Gly ThrAla Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro AlaSer Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn LysLeu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln ArgMet Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr TyrVal Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln GluVal Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile ProLeu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu AsnSer Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys ThrGly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 HisGly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155160 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165170 175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn CysGln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly ArgCys Arg 210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys AlaAla Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val CysArg Lys Phe Arg Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro ProLeu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn ProGlu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys ProArg Asn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala CysGly Ala Asp Ser Tyr Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg LysCys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly IleGly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr AsnIle Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu HisIle Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 ProPro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395400 Ile Thr Gly Leu Ser 405 11 2829 DNA EE(homo sapiens) CDS (1)..(2826)11 atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg gct 48Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 1015 gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag cgt aat tat gtg 96Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Arg Asn Tyr Val 20 25 30gtg aca gat cac ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat 144 ValThr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr 35 40 45 gagatg gag gaa gac ggc gtc cgc aag tgt aag aag tgc gaa ggg cct 192 Glu MetGlu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro 50 55 60 tgc cgcaaa gtg tgt aac gga ata ggt att ggt gaa ttt aaa gac tca 240 Cys Arg LysVal Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser 65 70 75 80 ctc tccata aat gct acg aat att aaa cac ttc aaa aac tgc acc tcc 288 Leu Ser IleAsn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser 85 90 95 atc agt ggcgat ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc 336 Ile Ser Gly AspLeu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser 100 105 110 ttc aca catact cct cct ctg gat cca cag gaa ctg gat att ctg aaa 384 Phe Thr His ThrPro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys 115 120 125 acc gta aaggaa atc aca ggg ttt ttg ctg att cag gct tgg cct gaa 432 Thr Val Lys GluIle Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu 130 135 140 aac agg acggac ctc cat gcc ttt gag aac cta gaa atc ata cgc ggc 480 Asn Arg Thr AspLeu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly 145 150 155 160 agg accaag caa cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac 528 Arg Thr LysGln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn 165 170 175 ata acatcc ttg gga tta cgc tcc ctc aag gag ata agt gat gga gat 576 Ile Thr SerLeu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp 180 185 190 gtg ataatt tca gga aac aaa aat ttg tgc tat gca aat aca ata aac 624 Val Ile IleSer Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn 195 200 205 tgg aaaaaa ctg ttt ggg acc tcc ggt cag aaa acc aaa att ata agc 672 Trp Lys LysLeu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser 210 215 220 aac agaggt gaa aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc 720 Asn Arg GlyGlu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala 225 230 235 240 ttgtgc tcc ccc gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc 768 Leu CysSer Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val 245 250 255 tcttgc cgg aat gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac 816 Ser CysArg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn 260 265 270 cttctg gag ggt gag cca agg gag ttt gtg gag aac tct gag tgc ata 864 Leu LeuGlu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile 275 280 285 cagtgc cac cca gag tgc ctg cct cag gcc atg aac atc acc tgc aca 912 Gln CysHis Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr 290 295 300 ggacgg gga cca gac aac tgt atc cag tgt gcc cac tac att gac ggc 960 Gly ArgGly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly 305 310 315 320ccc cac tgc gtc aag acc tgc ccg gca gga gtc atg gga gaa aac aac 1008 ProHis Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn 325 330 335acc ctg gtc tgg aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc 1056 ThrLeu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys 340 345 350cat cca aac tgc acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt 1104 HisPro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys 355 360 365cca acg aat ggg cct aag atc ccg tcc atc gcc act ggg atg gtg ggg 1152 ProThr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly 370 375 380gcc ctc ctc ttg ctg ctg gtg gtg gcc ctg ggg atc ggc ctc ttc atg 1200 AlaLeu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 385 390 395400 cga agg cgc cac atc gtt cgg aag cgc acg ctg cgg agg ctg ctg cag 1248Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln 405 410415 gag agg gag ctt gtg gag cct ctt aca ccc agt gga gaa gct ccc aac 1296Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn 420 425430 caa gct ctc ttg agg atc ttg aag gaa act gaa ttc aaa aag atc aaa 1344Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys 435 440445 gtg ctg ggc tcc ggt gcg ttc ggc acg gtg tat aag gga ctc tgg atc 1392Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile 450 455460 cca gaa ggt gag aaa gtt aaa att ccc gtc gct atc aag gaa tta aga 1440Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg 465 470475 480 gaa gca aca tct ccg aaa gcc aac aag gaa atc ctc gat gaa gcc tac1488 Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr 485490 495 gtg atg gcc agc gtg gac aac ccc cac gtg tgc cgc ctg ctg ggc atc1536 Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile 500505 510 tgc ctc acc tcc acc gtg cag ctc atc acg cag ctc atg ccc ttc ggc1584 Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly 515520 525 tgc ctc ctg gac tat gtc cgg gaa cac aaa gac aat att ggc tcc cag1632 Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln 530535 540 tac ctg ctc aac tgg tgt gtg cag atc gca aag ggc atg aac tac ttg1680 Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu 545550 555 560 gag gac cgt cgc ttg gtg cac cgc gac ctg gca gcc agg aac gtactg 1728 Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu565 570 575 gtg aaa aca ccg cag cat gtc aag atc aca gat ttt ggg ctg gccaaa 1776 Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys580 585 590 ctg ctg ggt gcg gaa gag aaa gaa tac cat gca gaa gga ggc aaagtg 1824 Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val595 600 605 cct atc aag tgg atg gca ttg gaa tca att tta cac aga atc tatacc 1872 Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr610 615 620 cac cag agt gat gtc tgg agc tac ggg gtg acc gtt tgg gag ttgatg 1920 His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met625 630 635 640 acc ttt gga tcc aag cca tat gac gga atc cct gcc agc gagatc tcc 1968 Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu IleSer 645 650 655 tcc atc ctg gag aaa gga gaa cgc ctc cct cag cca ccc atatgt acc 2016 Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile CysThr 660 665 670 atc gat gtc tac atg atc atg gtc aag tgc tgg atg ata gacgca gat 2064 Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp AlaAsp 675 680 685 agt cgc cca aag ttc cgt gag ttg atc atc gaa ttc tcc aaaatg gcc 2112 Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys MetAla 690 695 700 cga gac ccc cag cgc tac ctt gtc att cag ggg gat gaa agaatg cat 2160 Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg MetHis 705 710 715 720 ttg cca agt cct aca gac tcc aac ttc tac cgt gcc ctgatg gat gaa 2208 Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu MetAsp Glu 725 730 735 gaa gac atg gac gac gtg gtg gat gcc gac gag tac ctcatc cca cag 2256 Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu IlePro Gln 740 745 750 cag ggc ttc ttc agc agc ccc tcc acg tca cgg act cccctc ctg agc 2304 Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro LeuLeu Ser 755 760 765 tct ctg agt gca acc agc aac aat tcc acc gtg gct tgcatt gat aga 2352 Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys IleAsp Arg 770 775 780 aat ggg ctg caa agc tgt ccc atc aag gaa gac agc ttcttg cag cga 2400 Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe LeuGln Arg 785 790 795 800 tac agc tca gac ccc aca ggc gcc ttg act gag gacagc ata gac gac 2448 Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp SerIle Asp Asp 805 810 815 acc ttc ctc cca gtg cct gaa tac ata aac cag tccgtt ccc aaa agg 2496 Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser ValPro Lys Arg 820 825 830 ccc gct ggc tct gtg cag aat cct gtc tat cac aatcag cct ctg aac 2544 Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn GlnPro Leu Asn 835 840 845 ccc gcg ccc agc aga gac cca cac tac cag gac ccccac agc act gca 2592 Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro HisSer Thr Ala 850 855 860 gtg ggc aac ccc gag tat ctc aac act gtc cag cccacc tgt gtc aac 2640 Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro ThrCys Val Asn 865 870 875 880 agc aca ttc gac agc cct gcc cac tgg gcc cagaaa ggc agc cac caa 2688 Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln LysGly Ser His Gln 885 890 895 att agc ctg gac aac cct gac tac cag cag gacttc ttt ccc aag gaa 2736 Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp PhePhe Pro Lys Glu 900 905 910 gcc aag cca aat ggc atc ttt aag ggc tcc acagct gaa aat gca gaa 2784 Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr AlaGlu Asn Ala Glu 915 920 925 tac cta agg gtc gcg cca caa agc agt gaa tttatt gga gca tga 2829 Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile GlyAla 930 935 940 12 942 PRT Human(homo sapiens) 12 Met Arg Pro Ser GlyThr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys ProAla Ser Arg Ala Leu Glu Glu Lys Arg Asn Tyr Val 20 25 30 Val Thr Asp HisGly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr 35 40 45 Glu Met Glu GluAsp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro 50 55 60 Cys Arg Lys ValCys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser 65 70 75 80 Leu Ser IleAsn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser 85 90 95 Ile Ser GlyAsp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser 100 105 110 Phe ThrHis Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys 115 120 125 ThrVal Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu 130 135 140Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly 145 150155 160 Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn165 170 175 Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp GlyAsp 180 185 190 Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn ThrIle Asn 195 200 205 Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr LysIle Ile Ser 210 215 220 Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly GlnVal Cys His Ala 225 230 235 240 Leu Cys Ser Pro Glu Gly Cys Trp Gly ProGlu Pro Arg Asp Cys Val 245 250 255 Ser Cys Arg Asn Val Ser Arg Gly ArgGlu Cys Val Asp Lys Cys Asn 260 265 270 Leu Leu Glu Gly Glu Pro Arg GluPhe Val Glu Asn Ser Glu Cys Ile 275 280 285 Gln Cys His Pro Glu Cys LeuPro Gln Ala Met Asn Ile Thr Cys Thr 290 295 300 Gly Arg Gly Pro Asp AsnCys Ile Gln Cys Ala His Tyr Ile Asp Gly 305 310 315 320 Pro His Cys ValLys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn 325 330 335 Thr Leu ValTrp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys 340 345 350 His ProAsn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys 355 360 365 ProThr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly 370 375 380Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 385 390395 400 Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln405 410 415 Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala ProAsn 420 425 430 Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys LysIle Lys 435 440 445 Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys GlyLeu Trp Ile 450 455 460 Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala IleLys Glu Leu Arg 465 470 475 480 Glu Ala Thr Ser Pro Lys Ala Asn Lys GluIle Leu Asp Glu Ala Tyr 485 490 495 Val Met Ala Ser Val Asp Asn Pro HisVal Cys Arg Leu Leu Gly Ile 500 505 510 Cys Leu Thr Ser Thr Val Gln LeuIle Thr Gln Leu Met Pro Phe Gly 515 520 525 Cys Leu Leu Asp Tyr Val ArgGlu His Lys Asp Asn Ile Gly Ser Gln 530 535 540 Tyr Leu Leu Asn Trp CysVal Gln Ile Ala Lys Gly Met Asn Tyr Leu 545 550 555 560 Glu Asp Arg ArgLeu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu 565 570 575 Val Lys ThrPro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys 580 585 590 Leu LeuGly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val 595 600 605 ProIle Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr 610 615 620His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met 625 630635 640 Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser645 650 655 Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile CysThr 660 665 670 Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile AspAla Asp 675 680 685 Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe SerLys Met Ala 690 695 700 Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly AspGlu Arg Met His 705 710 715 720 Leu Pro Ser Pro Thr Asp Ser Asn Phe TyrArg Ala Leu Met Asp Glu 725 730 735 Glu Asp Met Asp Asp Val Val Asp AlaAsp Glu Tyr Leu Ile Pro Gln 740 745 750 Gln Gly Phe Phe Ser Ser Pro SerThr Ser Arg Thr Pro Leu Leu Ser 755 760 765 Ser Leu Ser Ala Thr Ser AsnAsn Ser Thr Val Ala Cys Ile Asp Arg 770 775 780 Asn Gly Leu Gln Ser CysPro Ile Lys Glu Asp Ser Phe Leu Gln Arg 785 790 795 800 Tyr Ser Ser AspPro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp 805 810 815 Thr Phe LeuPro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg 820 825 830 Pro AlaGly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn 835 840 845 ProAla Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala 850 855 860Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn 865 870875 880 Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln885 890 895 Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro LysGlu 900 905 910 Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu AsnAla Glu 915 920 925 Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile GlyAla 930 935 940 13 3633 DNA D-E>(Mus musculus) CDS (1)..(3630) 13 atgcga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc 48 Met ArgPro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr 1 5 10 15 gcgctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa 96 Ala LeuCys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 ggc acaagt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt 144 Gly Thr SerAsn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 ctg agc ctgcag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac 192 Leu Ser Leu GlnArg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 ttg gaa att acctat gtg caa agg aat tac gac ctt tcc ttc tta aag 240 Leu Glu Ile Thr TyrVal Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 acc atc cag gaggtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg 288 Thr Ile Gln Glu ValAla Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 gag aga atc cct ttggag aac ctg cag atc atc agg gga aat gct ctt 336 Glu Arg Ile Pro Leu GluAsn Leu Gln Ile Ile Arg Gly Asn Ala Leu 100 105 110 tat gaa aac acc tatgcc tta gcc atc ctg tcc aac tat ggg aca aac 384 Tyr Glu Asn Thr Tyr AlaLeu Ala Ile Leu Ser Asn Tyr Gly Thr Asn 115 120 125 aga act ggg ctt agggaa ctg ccc atg cgg aac tta cag gaa atc ctg 432 Arg Thr Gly Leu Arg GluLeu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 att ggt gct gtg cgattc agc aac aac ccc atc ctc tgc aat atg gat 480 Ile Gly Ala Val Arg PheSer Asn Asn Pro Ile Leu Cys Asn Met Asp 145 150 155 160 act atc cag tggagg gac atc gtc caa aac gtc ttt atg agc aac atg 528 Thr Ile Gln Trp ArgAsp Ile Val Gln Asn Val Phe Met Ser Asn Met 165 170 175 tca atg gac ttacag agc cat ccg agc agt tgc ccc aaa tgt gat cca 576 Ser Met Asp Leu GlnSer His Pro Ser Ser Cys Pro Lys Cys Asp Pro 180 185 190 agc tgt ccc aatgga agc tgc tgg gga gga gga gag gag aac tgc cag 624 Ser Cys Pro Asn GlySer Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln 195 200 205 aaa ttg acc aaaatc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt 672 Lys Leu Thr Lys IleIle Cys Ala Gln Gln Cys Ser His Arg Cys Arg 210 215 220 ggc agg tcc cccagt gac tgc tgc cac aac caa tgt gct gcg ggg tgt 720 Gly Arg Ser Pro SerAsp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 aca ggg ccccga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat 768 Thr Gly Pro ArgGlu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp 245 250 255 gag gcc acatgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc 816 Glu Ala Thr CysLys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 acc acc tatcag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt 864 Thr Thr Tyr GlnMet Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 gcc acc tgtgtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912 Ala Thr Cys ValLys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300 ggc tca tgtgtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960 Gly Ser Cys ValArg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu 305 310 315 320 gat ggcatc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008 Asp Gly IleArg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val 325 330 335 tgt aatggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056 Cys Asn GlyIle Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn 340 345 350 gct acaaac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104 Ala Thr AsnIle Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp 355 360 365 ctt cacatc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152 Leu His IleLeu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr 370 375 380 cct cctcta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200 Pro Pro LeuAsp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu 385 390 395 400 ataaca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248 Ile ThrGly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp 405 410 415 ctccat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296 Leu HisAla Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 catggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344 His GlyGln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu 435 440 445 gggctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392 Gly LeuArg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 ggaaac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440 Gly AsnArg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag 1488 PheGly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu 485 490 495aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536 LysAsp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser 500 505 510gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat 1584 GluGly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn 515 520 525gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632 ValSer Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly 530 535 540gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca 1680 GluPro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555560 gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570575 gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585590 aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtc tgg 1824Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp 595 600605 aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys 610 615620 acc tat gga tgt gct ggg cca ggt ctt caa gga tgt gaa gtg tgg cca 1920Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro 625 630635 640 tct ggg cca aag ata cca tct att gcc act ggg att gtg ggt ggc ctc1968 Ser Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Ile Val Gly Gly Leu 645650 655 ctc ttc ata gtg gtg gtg gcc ctt ggg att ggc cta ttc atg cga aga2016 Leu Phe Ile Val Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg 660665 670 cgt cac att gtt cga aag cgt aca cta cgc cgc ctg ctt caa gag aga2064 Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg 675680 685 gag ctc gtg gaa cct ctc aca ccc agc gga gaa gct cca aac caa gcc2112 Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala 690695 700 cac ttg agg ata tta aag gaa aca gaa ttc aaa aag atc aaa gtt ctg2160 His Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu 705710 715 720 ggt tcg gga gca ttt ggc aca gtg tat aag ggt ctc tgg atc ccagaa 2208 Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu725 730 735 ggt gag aaa gta aaa atc ccg gtg gcc atc aag gag tta aga gaagcc 2256 Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala740 745 750 aca tct cca aaa gcc aac aaa gaa atc ctt gac gaa gcc tat gtgatg 2304 Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met755 760 765 gct agt gtg gac aac cct cat gta tgc cgc ctc ctg ggc atc tgtctg 2352 Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu770 775 780 acc tcc act gtc cag ctc att aca cag ctc atg ccc tac ggt tgcctc 2400 Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys Leu785 790 795 800 ctg gac tac gtc cga gaa cac aag gac aac att ggc tcc cagtac ctc 2448 Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln TyrLeu 805 810 815 ctc aac tgg tgt gtg cag att gca aag ggc atg aac tac ctggaa gat 2496 Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu GluAsp 820 825 830 cgg cgt ttg gtg cac cgt gac ttg gca gcc agg aat gta ctggtg aag 2544 Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu ValLys 835 840 845 aca cca cag cat gtc aag atc aca gat ttt ggg ctg gcc aaactg ctt 2592 Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys LeuLeu 850 855 860 ggt gct gaa gag aaa gaa tat cat gcc gag ggg ggc aaa gtgcct atc 2640 Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val ProIle 865 870 875 880 aag tgg atg gct ttg gaa tca att tta cac cga att tataca cac caa 2688 Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr ThrHis Gln 885 890 895 agt gat gtc tgg agc tat ggt gtc act gtg tgg gaa ctgatg acc ttt 2736 Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu MetThr Phe 900 905 910 ggg tcc aag cct tat gat gga atc cca gca agt gac atctca tcc atc 2784 Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Asp Ile SerSer Ile 915 920 925 cta gag aaa gga gaa cgc ctt cca cag cca cct atc tgcacc atc gat 2832 Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys ThrIle Asp 930 935 940 gtc tac atg atc atg gtc aag tgc tgg atg ata gat gctgat agc cgc 2880 Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala AspSer Arg 945 950 955 960 cca aag ttc cga gag ttg att ctt gaa ttc tcc aaaatg gcc cga gac 2928 Pro Lys Phe Arg Glu Leu Ile Leu Glu Phe Ser Lys MetAla Arg Asp 965 970 975 cca cag cgc tac ctt gtt atc cag ggg gat gaa agaatg cat ttg cca 2976 Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg MetHis Leu Pro 980 985 990 agc cct aca gac tcc aac ttt tac cga gcc ctg atggat gaa gag gac 3024 Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met AspGlu Glu Asp 995 1000 1005 atg gag gat gta gtt gat gct gat gag tat cttacc cca cag caa 3069 Met Glu Asp Val Val Asp Ala Asp Glu Tyr Leu Thr ProGln Gln 1010 1015 1020 ggc ttc ttc aac agc ccg tcc acg tcg agg act cccctc ttg agt 3114 Gly Phe Phe Asn Ser Pro Ser Thr Ser Arg Thr Pro Leu LeuSer 1025 1030 1035 tct ctg agt gca act agc aac aat tcc act gtg gct tgcatt aat 3159 Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asn1040 1045 1050 aga aat ggg agc tgc cgt gtc aaa gaa gac gcc ttc ttg cagcgg 3204 Arg Asn Gly Ser Cys Arg Val Lys Glu Asp Ala Phe Leu Gln Arg1055 1060 1065 tac agc tcc gac ccc aca ggt gct gta aca gag gac aac atagat 3249 Tyr Ser Ser Asp Pro Thr Gly Ala Val Thr Glu Asp Asn Ile Asp1070 1075 1080 gac gca ttc ctc cct gta cct gaa tat gta aac caa tct gttccc 3294 Asp Ala Phe Leu Pro Val Pro Glu Tyr Val Asn Gln Ser Val Pro1085 1090 1095 aag agg cca gca ggc tct gtg cag aac cct gtc tat cac aatcag 3339 Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln1100 1105 1110 ccc ctg cat cca gct cct gga aga gac ctg cat tat caa aatccc 3384 Pro Leu His Pro Ala Pro Gly Arg Asp Leu His Tyr Gln Asn Pro1115 1120 1125 cac agc aat gca gtg ggc aac cct gag tat ctc aac act gcccag 3429 His Ser Asn Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Ala Gln1130 1135 1140 cct acc tgt ctc agt agt ggg ttt aac agc cct gca ctc tggatc 3474 Pro Thr Cys Leu Ser Ser Gly Phe Asn Ser Pro Ala Leu Trp Ile1145 1150 1155 cag aaa ggc agt cac caa atg agc cta gac aac cct gac taccag 3519 Gln Lys Gly Ser His Gln Met Ser Leu Asp Asn Pro Asp Tyr Gln1160 1165 1170 cag gac ttc ttc ccc aag gaa acc aag cca aat ggc ata tttaag 3564 Gln Asp Phe Phe Pro Lys Glu Thr Lys Pro Asn Gly Ile Phe Lys1175 1180 1185 ggc ccc aca gct gaa aat gca gag tac cta cgg gtg gca cctcca 3609 Gly Pro Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Pro1190 1195 1200 agc agc gag ttt att gga gca tga 3633 Ser Ser Glu Phe IleGly Ala 1205 1210 14 1210 PRT D-E>(Mus musculus) 14 Met Arg Pro Ser GlyThr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr 1 5 10 15 Ala Leu Cys AlaAla Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser AsnArg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu GlnArg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile ThrTyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile GlnGlu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg IlePro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu 100 105 110 Tyr GluAsn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn 115 120 125 ArgThr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp 145 150155 160 Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met165 170 175 Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys AspPro 180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu AsnCys Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser HisArg Cys Arg 210 215 220 Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln CysAla Ala Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu ValCys Gln Lys Phe Gln Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys ProPro Leu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val AsnPro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys CysPro Arg Asn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg AlaCys Gly Pro Asp Tyr Tyr Glu Val Glu Glu 305 310 315 320 Asp Gly Ile ArgLys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn GlyIle Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn 340 345 350 Ala ThrAsn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp 355 360 365 LeuHis Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr 370 375 380Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu 385 390395 400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp405 410 415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr LysGln 420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile ThrSer Leu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp ValIle Ile Ser 450 455 460 Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile AsnTrp Lys Lys Leu 465 470 475 480 Phe Gly Thr Pro Asn Gln Lys Thr Lys IleMet Asn Asn Arg Ala Glu 485 490 495 Lys Asp Cys Lys Ala Val Asn His ValCys Asn Pro Leu Cys Ser Ser 500 505 510 Glu Gly Cys Trp Gly Pro Glu ProArg Asp Cys Val Ser Cys Gln Asn 515 520 525 Val Ser Arg Gly Arg Glu CysVal Glu Lys Cys Asn Ile Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe ValGlu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys Leu ProGln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn CysIle Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys ThrCys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 LysTyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys 610 615 620Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro 625 630635 640 Ser Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Ile Val Gly Gly Leu645 650 655 Leu Phe Ile Val Val Val Ala Leu Gly Ile Gly Leu Phe Met ArgArg 660 665 670 Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu GlnGlu Arg 675 680 685 Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala ProAsn Gln Ala 690 695 700 His Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys LysIle Lys Val Leu 705 710 715 720 Gly Ser Gly Ala Phe Gly Thr Val Tyr LysGly Leu Trp Ile Pro Glu 725 730 735 Gly Glu Lys Val Lys Ile Pro Val AlaIle Lys Glu Leu Arg Glu Ala 740 745 750 Thr Ser Pro Lys Ala Asn Lys GluIle Leu Asp Glu Ala Tyr Val Met 755 760 765 Ala Ser Val Asp Asn Pro HisVal Cys Arg Leu Leu Gly Ile Cys Leu 770 775 780 Thr Ser Thr Val Gln LeuIle Thr Gln Leu Met Pro Tyr Gly Cys Leu 785 790 795 800 Leu Asp Tyr ValArg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu 805 810 815 Leu Asn TrpCys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp 820 825 830 Arg ArgLeu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys 835 840 845 ThrPro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu 850 855 860Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile 865 870875 880 Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln885 890 895 Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met ThrPhe 900 905 910 Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Asp Ile SerSer Ile 915 920 925 Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile CysThr Ile Asp 930 935 940 Val Tyr Met Ile Met Val Lys Cys Trp Met Ile AspAla Asp Ser Arg 945 950 955 960 Pro Lys Phe Arg Glu Leu Ile Leu Glu PheSer Lys Met Ala Arg Asp 965 970 975 Pro Gln Arg Tyr Leu Val Ile Gln GlyAsp Glu Arg Met His Leu Pro 980 985 990 Ser Pro Thr Asp Ser Asn Phe TyrArg Ala Leu Met Asp Glu Glu Asp 995 1000 1005 Met Glu Asp Val Val AspAla Asp Glu Tyr Leu Thr Pro Gln Gln 1010 1015 1020 Gly Phe Phe Asn SerPro Ser Thr Ser Arg Thr Pro Leu Leu Ser 1025 1030 1035 Ser Leu Ser AlaThr Ser Asn Asn Ser Thr Val Ala Cys Ile Asn 1040 1045 1050 Arg Asn GlySer Cys Arg Val Lys Glu Asp Ala Phe Leu Gln Arg 1055 1060 1065 Tyr SerSer Asp Pro Thr Gly Ala Val Thr Glu Asp Asn Ile Asp 1070 1075 1080 AspAla Phe Leu Pro Val Pro Glu Tyr Val Asn Gln Ser Val Pro 1085 1090 1095Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln 1100 11051110 Pro Leu His Pro Ala Pro Gly Arg Asp Leu His Tyr Gln Asn Pro 11151120 1125 His Ser Asn Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Ala Gln1130 1135 1140 Pro Thr Cys Leu Ser Ser Gly Phe Asn Ser Pro Ala Leu TrpIle 1145 1150 1155 Gln Lys Gly Ser His Gln Met Ser Leu Asp Asn Pro AspTyr Gln 1160 1165 1170 Gln Asp Phe Phe Pro Lys Glu Thr Lys Pro Asn GlyIle Phe Lys 1175 1180 1185 Gly Pro Thr Ala Glu Asn Ala Glu Tyr Leu ArgVal Ala Pro Pro 1190 1195 1200 Ser Ser Glu Phe Ile Gly Ala 1205 1210 151932 DNA D-E>(Mus musculus) CDS (1)..(1929) 15 atg cga ccc tca ggg accgcg aga acc aca ctg ctg gtg ctg ctg acc 48 Met Arg Pro Ser Gly Thr AlaArg Thr Thr Leu Leu Val Leu Leu Thr 1 5 10 15 gcg ctc tgc gcc gca ggtggg gcg ttg gag gaa aag aaa gtc tgc caa 96 Ala Leu Cys Ala Ala Gly GlyAla Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 ggc aca agt aac agg ctc acccaa ctg ggc act ttt gaa gac cac ttt 144 Gly Thr Ser Asn Arg Leu Thr GlnLeu Gly Thr Phe Glu Asp His Phe 35 40 45 ctg agc ctg cag agg atg tac aacaac tgt gaa gtg gtc ctt ggg aac 192 Leu Ser Leu Gln Arg Met Tyr Asn AsnCys Glu Val Val Leu Gly Asn 50 55 60 ttg gaa att acc tat gtg caa agg aattac gac ctt tcc ttc tta aag 240 Leu Glu Ile Thr Tyr Val Gln Arg Asn TyrAsp Leu Ser Phe Leu Lys 65 70 75 80 acc atc cag gag gtg gcc ggc tat gtcctc att gcc ctc aac acc gtg 288 Thr Ile Gln Glu Val Ala Gly Tyr Val LeuIle Ala Leu Asn Thr Val 85 90 95 gag aga atc cct ttg gag aac ctg cag atcatc agg gga aat gct ctt 336 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile IleArg Gly Asn Ala Leu 100 105 110 tat gaa aac acc tat gcc tta gcc atc ctgtcc aac tat ggg aca aac 384 Tyr Glu Asn Thr Tyr Ala Leu Ala Ile Leu SerAsn Tyr Gly Thr Asn 115 120 125 aga act ggg ctt agg gaa ctg ccc atg cggaac tta cag gaa atc ctg 432 Arg Thr Gly Leu Arg Glu Leu Pro Met Arg AsnLeu Gln Glu Ile Leu 130 135 140 att ggt gct gtg cga ttc agc aac aac cccatc ctc tgc aat atg gat 480 Ile Gly Ala Val Arg Phe Ser Asn Asn Pro IleLeu Cys Asn Met Asp 145 150 155 160 act atc cag tgg agg gac atc gtc caaaac gtc ttt atg agc aac atg 528 Thr Ile Gln Trp Arg Asp Ile Val Gln AsnVal Phe Met Ser Asn Met 165 170 175 tca atg gac tta cag agc cat ccg agcagt tgc ccc aaa tgt gat cca 576 Ser Met Asp Leu Gln Ser His Pro Ser SerCys Pro Lys Cys Asp Pro 180 185 190 agc tgt ccc aat gga agc tgc tgg ggagga gga gag gag aac tgc cag 624 Ser Cys Pro Asn Gly Ser Cys Trp Gly GlyGly Glu Glu Asn Cys Gln 195 200 205 aaa ttg acc aaa atc atc tgt gcc cagcaa tgt tcc cat cgc tgt cgt 672 Lys Leu Thr Lys Ile Ile Cys Ala Gln GlnCys Ser His Arg Cys Arg 210 215 220 ggc agg tcc ccc agt gac tgc tgc cacaac caa tgt gct gcg ggg tgt 720 Gly Arg Ser Pro Ser Asp Cys Cys His AsnGln Cys Ala Ala Gly Cys 225 230 235 240 aca ggg ccc cga gag agt gac tgtctg gtc tgc caa aag ttc caa gat 768 Thr Gly Pro Arg Glu Ser Asp Cys LeuVal Cys Gln Lys Phe Gln Asp 245 250 255 gag gcc aca tgc aaa gac acc tgccca cca ctc atg ctg tac aac ccc 816 Glu Ala Thr Cys Lys Asp Thr Cys ProPro Leu Met Leu Tyr Asn Pro 260 265 270 acc acc tat cag atg gat gtc aaccct gaa ggg aag tac agc ttt ggt 864 Thr Thr Tyr Gln Met Asp Val Asn ProGlu Gly Lys Tyr Ser Phe Gly 275 280 285 gcc acc tgt gtg aag aag tgc ccccga aac tac gtg gtg aca gat cat 912 Ala Thr Cys Val Lys Lys Cys Pro ArgAsn Tyr Val Val Thr Asp His 290 295 300 ggc tca tgt gtc cga gcc tgt gggcct gac tac tac gaa gtg gaa gaa 960 Gly Ser Cys Val Arg Ala Cys Gly ProAsp Tyr Tyr Glu Val Glu Glu 305 310 315 320 gat ggc atc cgc aag tgt aaaaaa tgt gat ggg ccc tgt cgc aaa gtt 1008 Asp Gly Ile Arg Lys Cys Lys LysCys Asp Gly Pro Cys Arg Lys Val 325 330 335 tgt aat ggc ata ggc att ggtgaa ttt aaa gac aca ctc tcc ata aat 1056 Cys Asn Gly Ile Gly Ile Gly GluPhe Lys Asp Thr Leu Ser Ile Asn 340 345 350 gct aca aac atc aaa cac ttcaaa tac tgc act gcc atc agc ggg gac 1104 Ala Thr Asn Ile Lys His Phe LysTyr Cys Thr Ala Ile Ser Gly Asp 355 360 365 ctt cac atc ctg cca gtg gccttt aag ggg gat tct ttc acg cgc act 1152 Leu His Ile Leu Pro Val Ala PheLys Gly Asp Ser Phe Thr Arg Thr 370 375 380 cct cct cta gac cca cga gaacta gaa att cta aaa acc gta aag gaa 1200 Pro Pro Leu Asp Pro Arg Glu LeuGlu Ile Leu Lys Thr Val Lys Glu 385 390 395 400 ata aca ggc ttt ttg ctgatt cag gct tgg cct gat aac tgg act gac 1248 Ile Thr Gly Phe Leu Leu IleGln Ala Trp Pro Asp Asn Trp Thr Asp 405 410 415 ctc cat gct ttc gag aaccta gaa ata ata cgt ggc aga aca aag caa 1296 Leu His Ala Phe Glu Asn LeuGlu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 cat ggt cag ttt tct ttggcg gtc gtt ggc ctg aac atc aca tca ctg 1344 His Gly Gln Phe Ser Leu AlaVal Val Gly Leu Asn Ile Thr Ser Leu 435 440 445 ggg ctg cgt tcc ctc aaggag atc agt gat ggg gat gtg atc att tct 1392 Gly Leu Arg Ser Leu Lys GluIle Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 gga aac cga aat ttg tgctac gca aac aca ata aac tgg aaa aaa ctc 1440 Gly Asn Arg Asn Leu Cys TyrAla Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 ttc ggg aca ccc aatcag aaa acc aaa atc atg aac aac aga gct gag 1488 Phe Gly Thr Pro Asn GlnLys Thr Lys Ile Met Asn Asn Arg Ala Glu 485 490 495 aaa gac tgc aag gccgtg aac cac gtc tgc aat cct tta tgc tcc tcg 1536 Lys Asp Cys Lys Ala ValAsn His Val Cys Asn Pro Leu Cys Ser Ser 500 505 510 gaa ggc tgc tgg ggccct gag ccc agg gac tgt gtc tcc tgc cag aat 1584 Glu Gly Cys Trp Gly ProGlu Pro Arg Asp Cys Val Ser Cys Gln Asn 515 520 525 gtg agc aga ggc agggag tgc gtg gag aaa tgc aac atc ctg gag ggg 1632 Val Ser Arg Gly Arg GluCys Val Glu Lys Cys Asn Ile Leu Glu Gly 530 535 540 gaa cca agg gag tttgtg gaa aat tct gaa tgc atc cag tgc cat cca 1680 Glu Pro Arg Glu Phe ValGlu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 gaa tgt ctg ccccag gcc atg aac atc acc tgt aca ggc agg ggg cca 1728 Glu Cys Leu Pro GlnAla Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 gac aac tgc atccag tgt gcc cac tac att gat ggc cca cac tgt gtc 1776 Asp Asn Cys Ile GlnCys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 aag acc tgc ccagct ggc atc atg gga gag aac aac act ctg gtc tgg 1824 Lys Thr Cys Pro AlaGly Ile Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 aag tat gca gatgcc aat aat gtc tgc cac cta tgc cac gcc aac tgt 1872 Lys Tyr Ala Asp AlaAsn Asn Val Cys His Leu Cys His Ala Asn Cys 610 615 620 acc tat gga gtaatg gtt cct gaa atg ttg ctc caa agt atc att tta 1920 Thr Tyr Gly Val MetVal Pro Glu Met Leu Leu Gln Ser Ile Ile Leu 625 630 635 640 aaa cct atttga 1932 Lys Pro Ile 16 643 PRT D-E>(Mus musculus) 16 Met Arg Pro SerGly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr 1 5 10 15 Ala Leu CysAla Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr SerAsn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser LeuGln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu IleThr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr IleGln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu ArgIle Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu 100 105 110 TyrGlu Asn Thr Tyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn 115 120 125Arg Thr Gly Leu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135140 Ile Gly Ala Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp 145150 155 160 Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser AsnMet 165 170 175 Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys CysAsp Pro 180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu GluAsn Cys Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys SerHis Arg Cys Arg 210 215 220 Gly Arg Ser Pro Ser Asp Cys Cys His Asn GlnCys Ala Ala Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys LeuVal Cys Gln Lys Phe Gln Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr CysPro Pro Leu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp ValAsn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys LysCys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val ArgAla Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu 305 310 315 320 Asp Gly IleArg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val 325 330 335 Cys AsnGly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn 340 345 350 AlaThr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp 355 360 365Leu His Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr 370 375380 Pro Pro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu 385390 395 400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp ThrAsp 405 410 415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg ThrLys Gln 420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn IleThr Ser Leu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly AspVal Ile Ile Ser 450 455 460 Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr IleAsn Trp Lys Lys Leu 465 470 475 480 Phe Gly Thr Pro Asn Gln Lys Thr LysIle Met Asn Asn Arg Ala Glu 485 490 495 Lys Asp Cys Lys Ala Val Asn HisVal Cys Asn Pro Leu Cys Ser Ser 500 505 510 Glu Gly Cys Trp Gly Pro GluPro Arg Asp Cys Val Ser Cys Gln Asn 515 520 525 Val Ser Arg Gly Arg GluCys Val Glu Lys Cys Asn Ile Leu Glu Gly 530 535 540 Glu Pro Arg Glu PheVal Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys LeuPro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp AsnCys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 LysThr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys 610 615620 Thr Tyr Gly Val Met Val Pro Glu Met Leu Leu Gln Ser Ile Ile Leu 625630 635 640 Lys Pro Ile 17 1968 DNA D-E (Mus musculus) CDS (1)..(1965)17 atg cga ccc tca ggg acc gcg aga acc aca ctg ctg gtg ctg ctg acc 48Met Arg Pro Ser Gly Thr Ala Arg Thr Thr Leu Leu Val Leu Leu Thr 1 5 1015 gcg ctc tgc gcc gca ggt ggg gcg ttg gag gaa aag aaa gtc tgc caa 96Ala Leu Cys Ala Ala Gly Gly Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30ggc aca agt aac agg ctc acc caa ctg ggc act ttt gaa gac cac ttt 144 GlyThr Ser Asn Arg Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 ctgagc ctg cag agg atg tac aac aac tgt gaa gtg gtc ctt ggg aac 192 Leu SerLeu Gln Arg Met Tyr Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 ttg gaaatt acc tat gtg caa agg aat tac gac ctt tcc ttc tta aag 240 Leu Glu IleThr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 acc atccag gag gtg gcc ggc tat gtc ctc att gcc ctc aac acc gtg 288 Thr Ile GlnGlu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 gag aga atccct ttg gag aac ctg cag atc atc agg gga aat gct ctt 336 Glu Arg Ile ProLeu Glu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu 100 105 110 tat gaa aacacc tat gcc tta gcc atc ctg tcc aac tat ggg aca aac 384 Tyr Glu Asn ThrTyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn 115 120 125 aga act gggctt agg gaa ctg ccc atg cgg aac tta cag gaa atc ctg 432 Arg Thr Gly LeuArg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 att ggt gctgtg cga ttc agc aac aac ccc atc ctc tgc aat atg gat 480 Ile Gly Ala ValArg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp 145 150 155 160 act atccag tgg agg gac atc gtc caa aac gtc ttt atg agc aac atg 528 Thr Ile GlnTrp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met 165 170 175 tca atggac tta cag agc cat ccg agc agt tgc ccc aaa tgt gat cca 576 Ser Met AspLeu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro 180 185 190 agc tgtccc aat gga agc tgc tgg gga gga gga gag gag aac tgc cag 624 Ser Cys ProAsn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln 195 200 205 aaa ttgacc aaa atc atc tgt gcc cag caa tgt tcc cat cgc tgt cgt 672 Lys Leu ThrLys Ile Ile Cys Ala Gln Gln Cys Ser His Arg Cys Arg 210 215 220 ggc aggtcc ccc agt gac tgc tgc cac aac caa tgt gct gcg ggg tgt 720 Gly Arg SerPro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 acaggg ccc cga gag agt gac tgt ctg gtc tgc caa aag ttc caa gat 768 Thr GlyPro Arg Glu Ser Asp Cys Leu Val Cys Gln Lys Phe Gln Asp 245 250 255 gaggcc aca tgc aaa gac acc tgc cca cca ctc atg ctg tac aac ccc 816 Glu AlaThr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 accacc tat cag atg gat gtc aac cct gaa ggg aag tac agc ttt ggt 864 Thr ThrTyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 gccacc tgt gtg aag aag tgc ccc cga aac tac gtg gtg aca gat cat 912 Ala ThrCys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300 ggctca tgt gtc cga gcc tgt ggg cct gac tac tac gaa gtg gaa gaa 960 Gly SerCys Val Arg Ala Cys Gly Pro Asp Tyr Tyr Glu Val Glu Glu 305 310 315 320gat ggc atc cgc aag tgt aaa aaa tgt gat ggg ccc tgt cgc aaa gtt 1008 AspGly Ile Arg Lys Cys Lys Lys Cys Asp Gly Pro Cys Arg Lys Val 325 330 335tgt aat ggc ata ggc att ggt gaa ttt aaa gac aca ctc tcc ata aat 1056 CysAsn Gly Ile Gly Ile Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn 340 345 350gct aca aac atc aaa cac ttc aaa tac tgc act gcc atc agc ggg gac 1104 AlaThr Asn Ile Lys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp 355 360 365ctt cac atc ctg cca gtg gcc ttt aag ggg gat tct ttc acg cgc act 1152 LeuHis Ile Leu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr 370 375 380cct cct cta gac cca cga gaa cta gaa att cta aaa acc gta aag gaa 1200 ProPro Leu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu 385 390 395400 ata aca ggc ttt ttg ctg att cag gct tgg cct gat aac tgg act gac 1248Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp 405 410415 ctc cat gct ttc gag aac cta gaa ata ata cgt ggc aga aca aag caa 1296Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425430 cat ggt cag ttt tct ttg gcg gtc gtt ggc ctg aac atc aca tca ctg 1344His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu 435 440445 ggg ctg cgt tcc ctc aag gag atc agt gat ggg gat gtg atc att tct 1392Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455460 gga aac cga aat ttg tgc tac gca aac aca ata aac tgg aaa aaa ctc 1440Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470475 480 ttc ggg aca ccc aat cag aaa acc aaa atc atg aac aac aga gct gag1488 Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met Asn Asn Arg Ala Glu 485490 495 aaa gac tgc aag gcc gtg aac cac gtc tgc aat cct tta tgc tcc tcg1536 Lys Asp Cys Lys Ala Val Asn His Val Cys Asn Pro Leu Cys Ser Ser 500505 510 gaa ggc tgc tgg ggc cct gag ccc agg gac tgt gtc tcc tgc cag aat1584 Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Gln Asn 515520 525 gtg agc aga ggc agg gag tgc gtg gag aaa tgc aac atc ctg gag ggg1632 Val Ser Arg Gly Arg Glu Cys Val Glu Lys Cys Asn Ile Leu Glu Gly 530535 540 gaa cca agg gag ttt gtg gaa aat tct gaa tgc atc cag tgc cat cca1680 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545550 555 560 gaa tgt ctg ccc cag gcc atg aac atc acc tgt aca ggc agg gggcca 1728 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575 gac aac tgc atc cag tgt gcc cac tac att gat ggc cca cac tgtgtc 1776 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585 590 aag acc tgc cca gct ggc atc atg gga gag aac aac act ctg gtctgg 1824 Lys Thr Cys Pro Ala Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605 aag tat gca gat gcc aat aat gtc tgc cac cta tgc cac gcc aactgt 1872 Lys Tyr Ala Asp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys610 615 620 acc tat gga tgt gct ggg cca ggt ctt caa gga tgt gaa gtg tggcca 1920 Thr Tyr Gly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro625 630 635 640 tct ggg tac gtt caa tgg cag tgg atc tta aag acc ttt tggatc taa 1968 Ser Gly Tyr Val Gln Trp Gln Trp Ile Leu Lys Thr Phe Trp Ile645 650 655 18 655 PRT D-E (Mus musculus) 18 Met Arg Pro Ser Gly Thr AlaArg Thr Thr Leu Leu Val Leu Leu Thr 1 5 10 15 Ala Leu Cys Ala Ala GlyGly Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn Arg LeuThr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln Arg MetTyr Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr Tyr ValGln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu ValAla Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro LeuGlu Asn Leu Gln Ile Ile Arg Gly Asn Ala Leu 100 105 110 Tyr Glu Asn ThrTyr Ala Leu Ala Ile Leu Ser Asn Tyr Gly Thr Asn 115 120 125 Arg Thr GlyLeu Arg Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 Ile GlyAla Val Arg Phe Ser Asn Asn Pro Ile Leu Cys Asn Met Asp 145 150 155 160Thr Ile Gln Trp Arg Asp Ile Val Gln Asn Val Phe Met Ser Asn Met 165 170175 Ser Met Asp Leu Gln Ser His Pro Ser Ser Cys Pro Lys Cys Asp Pro 180185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Gly Gly Glu Glu Asn Cys Gln195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser His Arg CysArg 210 215 220 Gly Arg Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala AlaGly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys GlnLys Phe Gln Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro LeuMet Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn Pro GluGly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys Pro ArgAsn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala Cys GlyPro Asp Tyr Tyr Glu Val Glu Glu 305 310 315 320 Asp Gly Ile Arg Lys CysLys Lys Cys Asp Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile GlyIle Gly Glu Phe Lys Asp Thr Leu Ser Ile Asn 340 345 350 Ala Thr Asn IleLys His Phe Lys Tyr Cys Thr Ala Ile Ser Gly Asp 355 360 365 Leu His IleLeu Pro Val Ala Phe Lys Gly Asp Ser Phe Thr Arg Thr 370 375 380 Pro ProLeu Asp Pro Arg Glu Leu Glu Ile Leu Lys Thr Val Lys Glu 385 390 395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Asp Asn Trp Thr Asp 405 410415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420425 430 His Gly Gln Phe Ser Leu Ala Val Val Gly Leu Asn Ile Thr Ser Leu435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile IleSer 450 455 460 Gly Asn Arg Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp LysLys Leu 465 470 475 480 Phe Gly Thr Pro Asn Gln Lys Thr Lys Ile Met AsnAsn Arg Ala Glu 485 490 495 Lys Asp Cys Lys Ala Val Asn His Val Cys AsnPro Leu Cys Ser Ser 500 505 510 Glu Gly Cys Trp Gly Pro Glu Pro Arg AspCys Val Ser Cys Gln Asn 515 520 525 Val Ser Arg Gly Arg Glu Cys Val GluLys Cys Asn Ile Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe Val Glu AsnSer Glu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys Leu Pro Gln AlaMet Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys Ile GlnCys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys Thr Cys ProAla Gly Ile Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys Tyr AlaAsp Ala Asn Asn Val Cys His Leu Cys His Ala Asn Cys 610 615 620 Thr TyrGly Cys Ala Gly Pro Gly Leu Gln Gly Cys Glu Val Trp Pro 625 630 635 640Ser Gly Tyr Val Gln Trp Gln Trp Ile Leu Lys Thr Phe Trp Ile 645 650 65519 1962 DNA 1/4Y CDS (1)..(1962) 19 atg ggt gta cgc agc ccc ctg tcc gcctct ggg cct cgc ggg gcc gct 48 Met Gly Val Arg Ser Pro Leu Ser Ala SerGly Pro Arg Gly Ala Ala 1 5 10 15 gtc ctg gtg cta ctg ctg ctg ggc gtcgcg ctg tgt tcc gcc gtg gag 96 Val Leu Val Leu Leu Leu Leu Gly Val AlaLeu Cys Ser Ala Val Glu 20 25 30 gag aag aaa gtt tgt caa ggg aca aat aacaag ttg acc cag ctg ggg 144 Glu Lys Lys Val Cys Gln Gly Thr Asn Asn LysLeu Thr Gln Leu Gly 35 40 45 cac gtg gaa gac cat ttc acc agc ctg cag agaatg tac aac aac tgc 192 His Val Glu Asp His Phe Thr Ser Leu Gln Arg MetTyr Asn Asn Cys 50 55 60 gaa gtg gta ctg agt aac ctg gag att acc tac gtggag cat aat cgc 240 Glu Val Val Leu Ser Asn Leu Glu Ile Thr Tyr Val GluHis Asn Arg 65 70 75 80 gat ctc acc ttc ctt aag acc ata cag gag gtt gcaggc tat gtg ctc 288 Asp Leu Thr Phe Leu Lys Thr Ile Gln Glu Val Ala GlyTyr Val Leu 85 90 95 att gcg ctt aac atg gtg gac gtc att ccc tta gaa aacctc cag att 336 Ile Ala Leu Asn Met Val Asp Val Ile Pro Leu Glu Asn LeuGln Ile 100 105 110 atc cga ggg aat gtg ctt tat gac aac tct ttt gcc ctggca gtt tta 384 Ile Arg Gly Asn Val Leu Tyr Asp Asn Ser Phe Ala Leu AlaVal Leu 115 120 125 tcc aat tac cac atg aat aaa acc cag gga ctt cga gagctg cca atg 432 Ser Asn Tyr His Met Asn Lys Thr Gln Gly Leu Arg Glu LeuPro Met 130 135 140 aaa cgg cta tca gaa att ctc aat gga ggt gtt aaa atcagc aac aac 480 Lys Arg Leu Ser Glu Ile Leu Asn Gly Gly Val Lys Ile SerAsn Asn 145 150 155 160 ccc aaa ctg tgc aac atg gac act gtt ctc tgg aatgac atc att gat 528 Pro Lys Leu Cys Asn Met Asp Thr Val Leu Trp Asn AspIle Ile Asp 165 170 175 aca agc agg aag cct ctc aca gta ctt gac ttt gcaagc aat ctt tct 576 Thr Ser Arg Lys Pro Leu Thr Val Leu Asp Phe Ala SerAsn Leu Ser 180 185 190 tct tgt cca aaa tgc cat ccg aac tgc aca gaa gaccac tgc tgg ggt 624 Ser Cys Pro Lys Cys His Pro Asn Cys Thr Glu Asp HisCys Trp Gly 195 200 205 gct ggt gaa cag aac tgc cag act tta aca aaa gtcatc tgt gcc cag 672 Ala Gly Glu Gln Asn Cys Gln Thr Leu Thr Lys Val IleCys Ala Gln 210 215 220 caa tgc tct ggc cgg tgc aga gga aag gtg ccc agtgac tgc tgc cac 720 Gln Cys Ser Gly Arg Cys Arg Gly Lys Val Pro Ser AspCys Cys His 225 230 235 240 aat cag tgt gct gca ggg tgc aca gga cct cgggag agt gac tgc ctg 768 Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg GluSer Asp Cys Leu 245 250 255 gca tgc cgc aag ttt cgg gat gat gct acc tgcaag gac aca tgt ccc 816 Ala Cys Arg Lys Phe Arg Asp Asp Ala Thr Cys LysAsp Thr Cys Pro 260 265 270 cca ctg gtc ctc tat aac ccc acc acc tat caaatg gat gtc aac cct 864 Pro Leu Val Leu Tyr Asn Pro Thr Thr Tyr Gln MetAsp Val Asn Pro 275 280 285 gag gga aaa tac agc ttt gga gcc act tgt gtgagg gaa tgt cca cac 912 Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val ArgGlu Cys Pro His 290 295 300 aac tat gtg gtg aca gat cat ggc tcc tgc gttcgc tcg tgt aat act 960 Asn Tyr Val Val Thr Asp His Gly Ser Cys Val ArgSer Cys Asn Thr 305 310 315 320 gat act tac gaa gtg gaa gaa aat ggt gttcgg aag tgt aaa aaa tgt 1008 Asp Thr Tyr Glu Val Glu Glu Asn Gly Val ArgLys Cys Lys Lys Cys 325 330 335 gat ggg cta tgt agc aaa gtg tgc aat ggcatt gga ata ggt gaa ctt 1056 Asp Gly Leu Cys Ser Lys Val Cys Asn Gly IleGly Ile Gly Glu Leu 340 345 350 aaa ggg atc cta tcc ata aat gcc aca aacatc gac tcc ttc aaa aac 1104 Lys Gly Ile Leu Ser Ile Asn Ala Thr Asn IleAsp Ser Phe Lys Asn 355 360 365 tgt acg aag atc aat ggg gat gtc agc attctt cct gtt gca ttt cta 1152 Cys Thr Lys Ile Asn Gly Asp Val Ser Ile LeuPro Val Ala Phe Leu 370 375 380 ggg gat gcc ttc aca aag aca cta ccc cttgac cct aag aag ctg gat 1200 Gly Asp Ala Phe Thr Lys Thr Leu Pro Leu AspPro Lys Lys Leu Asp 385 390 395 400 gtc ttt aga aca gtc aaa gaa ata tcagga ttt ttg ttg att cag gcc 1248 Val Phe Arg Thr Val Lys Glu Ile Ser GlyPhe Leu Leu Ile Gln Ala 405 410 415 tgg cct gat aat gct act gat ctc tatgct ttt gaa aat ctg gag att 1296 Trp Pro Asp Asn Ala Thr Asp Leu Tyr AlaPhe Glu Asn Leu Glu Ile 420 425 430 atc cga ggc cga acc aag cag cac ggccag tat tcc ctt gct gtt gtt 1344 Ile Arg Gly Arg Thr Lys Gln His Gly GlnTyr Ser Leu Ala Val Val 435 440 445 aac ttg aaa ata cag tcg ttg ggg ctgcgc tcc ctc aag gaa ata agt 1392 Asn Leu Lys Ile Gln Ser Leu Gly Leu ArgSer Leu Lys Glu Ile Ser 450 455 460 gat gga gac att gcc att atg aag aacaag aac ctc tgc tat gct gac 1440 Asp Gly Asp Ile Ala Ile Met Lys Asn LysAsn Leu Cys Tyr Ala Asp 465 470 475 480 acc atg aac tgg cgc agc ttg tttgct act cag agt cag aaa aca aaa 1488 Thr Met Asn Trp Arg Ser Leu Phe AlaThr Gln Ser Gln Lys Thr Lys 485 490 495 att ata cag aac aga aat aaa aatgat tgt act gct gac agg cat gtg 1536 Ile Ile Gln Asn Arg Asn Lys Asn AspCys Thr Ala Asp Arg His Val 500 505 510 tgt gac ccg ctg tgc tcg gac gtgggc tgc tgg ggc cca ggg ccc ttc 1584 Cys Asp Pro Leu Cys Ser Asp Val GlyCys Trp Gly Pro Gly Pro Phe 515 520 525 cac tgc ttt tcc tgc agg ttt ttcagt cgc cag aag gag tgt gta aaa 1632 His Cys Phe Ser Cys Arg Phe Phe SerArg Gln Lys Glu Cys Val Lys 530 535 540 cag tgc aac atc ctg caa ggg gagcca cgt gag ttt gaa aga gac tcc 1680 Gln Cys Asn Ile Leu Gln Gly Glu ProArg Glu Phe Glu Arg Asp Ser 545 550 555 560 aaa tgc ctc ccc tgc cac tcagag tgt ctg gta cag aac tcc act gca 1728 Lys Cys Leu Pro Cys His Ser GluCys Leu Val Gln Asn Ser Thr Ala 565 570 575 tac aac aca acc tgc tct ggaccg ggc cca gac cac tgc atg aag tgt 1776 Tyr Asn Thr Thr Cys Ser Gly ProGly Pro Asp His Cys Met Lys Cys 580 585 590 gcc cat ttt ata gat ggt ccccac tgt gtg aag gcc tgc ccc gct ggg 1824 Ala His Phe Ile Asp Gly Pro HisCys Val Lys Ala Cys Pro Ala Gly 595 600 605 gtc ctg ggt gag aat gat accctg gtc tgg aag tat gca gat gcc aat 1872 Val Leu Gly Glu Asn Asp Thr LeuVal Trp Lys Tyr Ala Asp Ala Asn 610 615 620 gct gtt tgc cag ctc tgc catcca aac tgt aca cga ggg tgc aaa ggg 1920 Ala Val Cys Gln Leu Cys His ProAsn Cys Thr Arg Gly Cys Lys Gly 625 630 635 640 cca ggt ctt gaa gga tgtcca aat ggc tcc aaa act cca tct 1962 Pro Gly Leu Glu Gly Cys Pro Asn GlySer Lys Thr Pro Ser 645 650 20 654 PRT 1/4Y 20 Met Gly Val Arg Ser ProLeu Ser Ala Ser Gly Pro Arg Gly Ala Ala 1 5 10 15 Val Leu Val Leu LeuLeu Leu Gly Val Ala Leu Cys Ser Ala Val Glu 20 25 30 Glu Lys Lys Val CysGln Gly Thr Asn Asn Lys Leu Thr Gln Leu Gly 35 40 45 His Val Glu Asp HisPhe Thr Ser Leu Gln Arg Met Tyr Asn Asn Cys 50 55 60 Glu Val Val Leu SerAsn Leu Glu Ile Thr Tyr Val Glu His Asn Arg 65 70 75 80 Asp Leu Thr PheLeu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu 85 90 95 Ile Ala Leu AsnMet Val Asp Val Ile Pro Leu Glu Asn Leu Gln Ile 100 105 110 Ile Arg GlyAsn Val Leu Tyr Asp Asn Ser Phe Ala Leu Ala Val Leu 115 120 125 Ser AsnTyr His Met Asn Lys Thr Gln Gly Leu Arg Glu Leu Pro Met 130 135 140 LysArg Leu Ser Glu Ile Leu Asn Gly Gly Val Lys Ile Ser Asn Asn 145 150 155160 Pro Lys Leu Cys Asn Met Asp Thr Val Leu Trp Asn Asp Ile Ile Asp 165170 175 Thr Ser Arg Lys Pro Leu Thr Val Leu Asp Phe Ala Ser Asn Leu Ser180 185 190 Ser Cys Pro Lys Cys His Pro Asn Cys Thr Glu Asp His Cys TrpGly 195 200 205 Ala Gly Glu Gln Asn Cys Gln Thr Leu Thr Lys Val Ile CysAla Gln 210 215 220 Gln Cys Ser Gly Arg Cys Arg Gly Lys Val Pro Ser AspCys Cys His 225 230 235 240 Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro ArgGlu Ser Asp Cys Leu 245 250 255 Ala Cys Arg Lys Phe Arg Asp Asp Ala ThrCys Lys Asp Thr Cys Pro 260 265 270 Pro Leu Val Leu Tyr Asn Pro Thr ThrTyr Gln Met Asp Val Asn Pro 275 280 285 Glu Gly Lys Tyr Ser Phe Gly AlaThr Cys Val Arg Glu Cys Pro His 290 295 300 Asn Tyr Val Val Thr Asp HisGly Ser Cys Val Arg Ser Cys Asn Thr 305 310 315 320 Asp Thr Tyr Glu ValGlu Glu Asn Gly Val Arg Lys Cys Lys Lys Cys 325 330 335 Asp Gly Leu CysSer Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Leu 340 345 350 Lys Gly IleLeu Ser Ile Asn Ala Thr Asn Ile Asp Ser Phe Lys Asn 355 360 365 Cys ThrLys Ile Asn Gly Asp Val Ser Ile Leu Pro Val Ala Phe Leu 370 375 380 GlyAsp Ala Phe Thr Lys Thr Leu Pro Leu Asp Pro Lys Lys Leu Asp 385 390 395400 Val Phe Arg Thr Val Lys Glu Ile Ser Gly Phe Leu Leu Ile Gln Ala 405410 415 Trp Pro Asp Asn Ala Thr Asp Leu Tyr Ala Phe Glu Asn Leu Glu Ile420 425 430 Ile Arg Gly Arg Thr Lys Gln His Gly Gln Tyr Ser Leu Ala ValVal 435 440 445 Asn Leu Lys Ile Gln Ser Leu Gly Leu Arg Ser Leu Lys GluIle Ser 450 455 460 Asp Gly Asp Ile Ala Ile Met Lys Asn Lys Asn Leu CysTyr Ala Asp 465 470 475 480 Thr Met Asn Trp Arg Ser Leu Phe Ala Thr GlnSer Gln Lys Thr Lys 485 490 495 Ile Ile Gln Asn Arg Asn Lys Asn Asp CysThr Ala Asp Arg His Val 500 505 510 Cys Asp Pro Leu Cys Ser Asp Val GlyCys Trp Gly Pro Gly Pro Phe 515 520 525 His Cys Phe Ser Cys Arg Phe PheSer Arg Gln Lys Glu Cys Val Lys 530 535 540 Gln Cys Asn Ile Leu Gln GlyGlu Pro Arg Glu Phe Glu Arg Asp Ser 545 550 555 560 Lys Cys Leu Pro CysHis Ser Glu Cys Leu Val Gln Asn Ser Thr Ala 565 570 575 Tyr Asn Thr ThrCys Ser Gly Pro Gly Pro Asp His Cys Met Lys Cys 580 585 590 Ala His PheIle Asp Gly Pro His Cys Val Lys Ala Cys Pro Ala Gly 595 600 605 Val LeuGly Glu Asn Asp Thr Leu Val Trp Lys Tyr Ala Asp Ala Asn 610 615 620 AlaVal Cys Gln Leu Cys His Pro Asn Cys Thr Arg Gly Cys Lys Gly 625 630 635640 Pro Gly Leu Glu Gly Cys Pro Asn Gly Ser Lys Thr Pro Ser 645 650 214134 DNA 1-O(Drosophila melanogaster) CDS (1)..(3630) 21 atg atg att atcagc atg tgg atg agc ata tcg cga gga ttg tgg gac 48 Met Met Ile Ile SerMet Trp Met Ser Ile Ser Arg Gly Leu Trp Asp 1 5 10 15 agc agc tcc atcttg tcg gtg ctg ctg atc ctc gcc tgc atg gca tcc 96 Ser Ser Ser Ile LeuSer Val Leu Leu Ile Leu Ala Cys Met Ala Ser 20 25 30 atc acc aca agc tcatcg gtc agc aat gcc ggc tat gtg gat aat ggc 144 Ile Thr Thr Ser Ser SerVal Ser Asn Ala Gly Tyr Val Asp Asn Gly 35 40 45 aat atg aaa gtc tgc atcggc act aaa tct cgg ctc tcc gtg ccc tcc 192 Asn Met Lys Val Cys Ile GlyThr Lys Ser Arg Leu Ser Val Pro Ser 50 55 60 aac aag gaa cat cat tac cgaaac ctc aga gat cgg tac acg aac tgt 240 Asn Lys Glu His His Tyr Arg AsnLeu Arg Asp Arg Tyr Thr Asn Cys 65 70 75 80 acg tat gtg gat ggc aac ttgaaa ctg acc tgg cta ccc aac gag aat 288 Thr Tyr Val Asp Gly Asn Leu LysLeu Thr Trp Leu Pro Asn Glu Asn 85 90 95 ttg gac ctc agc ttc cta gac aacata cgg gag gtc acc ggc tat att 336 Leu Asp Leu Ser Phe Leu Asp Asn IleArg Glu Val Thr Gly Tyr Ile 100 105 110 ctg atc agt cat gtg gac gtt aagaaa gtg gtg ttt ccc aaa cta caa 384 Leu Ile Ser His Val Asp Val Lys LysVal Val Phe Pro Lys Leu Gln 115 120 125 atc att cgc gga cgc acg ctg ttcagc tta tcc gtg gag gag gag aag 432 Ile Ile Arg Gly Arg Thr Leu Phe SerLeu Ser Val Glu Glu Glu Lys 130 135 140 tat gcc ttg ttc gtc act tat tccaaa atg tac acg ctg gag att ccc 480 Tyr Ala Leu Phe Val Thr Tyr Ser LysMet Tyr Thr Leu Glu Ile Pro 145 150 155 160 gat cta cgc gat gtc tta aatggc caa gtg ggc ttc cac aac aac tac 528 Asp Leu Arg Asp Val Leu Asn GlyGln Val Gly Phe His Asn Asn Tyr 165 170 175 aat ctc tgc cac atg cga acgatc cag tgg tcg gag att gta tcc aac 576 Asn Leu Cys His Met Arg Thr IleGln Trp Ser Glu Ile Val Ser Asn 180 185 190 ggc acg gat gca tac tac aactac gac ttt act gct ccg gag cgc gag 624 Gly Thr Asp Ala Tyr Tyr Asn TyrAsp Phe Thr Ala Pro Glu Arg Glu 195 200 205 tgt ccc aag tgc cac gag agctgc acg cac gga tgt tgg ggc gag ggt 672 Cys Pro Lys Cys His Glu Ser CysThr His Gly Cys Trp Gly Glu Gly 210 215 220 ccc aag aat tgc cag aag ttcagc aag ctc acc tgc tcg cca cag tgt 720 Pro Lys Asn Cys Gln Lys Phe SerLys Leu Thr Cys Ser Pro Gln Cys 225 230 235 240 gcc gga ggt cgt tgc tatgga cca aag ccg cgg gag tgt tgt cac ctc 768 Ala Gly Gly Arg Cys Tyr GlyPro Lys Pro Arg Glu Cys Cys His Leu 245 250 255 ttc tgc gcc gga gga tgcact ggt ccc acg caa aag gat tgc atc gcc 816 Phe Cys Ala Gly Gly Cys ThrGly Pro Thr Gln Lys Asp Cys Ile Ala 260 265 270 tgc aag aac ttc ttc gacgag gca gta tca aag gag gaa tgc ccg ccc 864 Cys Lys Asn Phe Phe Asp GluAla Val Ser Lys Glu Glu Cys Pro Pro 275 280 285 atg cgc aag tac aat cccacc acc tat gtt ctt gaa acg aat cct gag 912 Met Arg Lys Tyr Asn Pro ThrThr Tyr Val Leu Glu Thr Asn Pro Glu 290 295 300 gga aag tat gcc tat ggtgcc acc tgc gtc aag gag tgt ccc ggt cat 960 Gly Lys Tyr Ala Tyr Gly AlaThr Cys Val Lys Glu Cys Pro Gly His 305 310 315 320 ctg ttg cgg gat aatggc gcc tgc gtg cgc agc tgt ccc cag gac aag 1008 Leu Leu Arg Asp Asn GlyAla Cys Val Arg Ser Cys Pro Gln Asp Lys 325 330 335 atg gac aag ggg ggcgag tgt gtg ccc tgc aat gga ccg tgc ccc aaa 1056 Met Asp Lys Gly Gly GluCys Val Pro Cys Asn Gly Pro Cys Pro Lys 340 345 350 acc tgc ccg ggc gttact gtc ctg cat gcc ggc aac att gac tcg ttc 1104 Thr Cys Pro Gly Val ThrVal Leu His Ala Gly Asn Ile Asp Ser Phe 355 360 365 cgg aat tgt acg gtgatc gat ggc aac att cgc att ttg gat cag acc 1152 Arg Asn Cys Thr Val IleAsp Gly Asn Ile Arg Ile Leu Asp Gln Thr 370 375 380 ttc tcg ggc ttc caggat gtc tat gcc aac tac acg atg gga cca cga 1200 Phe Ser Gly Phe Gln AspVal Tyr Ala Asn Tyr Thr Met Gly Pro Arg 385 390 395 400 tac ata ccg ctggat ccc gag cga cgg gag gtg ttc tcc acg gtg aag 1248 Tyr Ile Pro Leu AspPro Glu Arg Arg Glu Val Phe Ser Thr Val Lys 405 410 415 gag atc acc gggtat ctg aat atc gag gga acc cac ccg cag ttc cgg 1296 Glu Ile Thr Gly TyrLeu Asn Ile Glu Gly Thr His Pro Gln Phe Arg 420 425 430 aat ctg tcg tacttt cgc aat ctg gaa aca att cat ggc cgc cag ctg 1344 Asn Leu Ser Tyr PheArg Asn Leu Glu Thr Ile His Gly Arg Gln Leu 435 440 445 atg gag agc atgttt gcc gct ttg gcg atc gtt aag tca tcc ctg tac 1392 Met Glu Ser Met PheAla Ala Leu Ala Ile Val Lys Ser Ser Leu Tyr 450 455 460 agc ctg gag atgcgc aat ctg aag cag att agt tcc ggc agt gtg gtc 1440 Ser Leu Glu Met ArgAsn Leu Lys Gln Ile Ser Ser Gly Ser Val Val 465 470 475 480 atc cag cataat aga gac ctc tgc tac gta agc aat atc cgt tgg ccg 1488 Ile Gln His AsnArg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp Pro 485 490 495 gcc att cagaag gag ccc gaa cag aag gtg tgg gtc aac gag aat ctc 1536 Ala Ile Gln LysGlu Pro Glu Gln Lys Val Trp Val Asn Glu Asn Leu 500 505 510 agg gcg gatcta tgc gag aaa aat gga acc att tgc tcg gat cag tgc 1584 Arg Ala Asp LeuCys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln Cys 515 520 525 aac gag gacggc tgc tgg gga gct ggc acg gat cag tgc ctt acc tgc 1632 Asn Glu Asp GlyCys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr Cys 530 535 540 aag aac ttcaat ttc aat ggc acc tgc atc gcc gac tgt ggt tat ata 1680 Lys Asn Phe AsnPhe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr Ile 545 550 555 560 tcc aatgcc tac aag ttt gac aat aga acg tgc aag ata tgc cat cca 1728 Ser Asn AlaTyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His Pro 565 570 575 gag tgccgg act tgc aat gga gct gga gca gat cac tgc cag gag tgc 1776 Glu Cys ArgThr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu Cys 580 585 590 gtc catgtg agg gac ggt cag cac tgt gtg tcc gag tgc ccg aag aac 1824 Val His ValArg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys Asn 595 600 605 aag tacaac gat cgt ggt gtc tgc cga gag tgc cac gcc acc tgc gat 1872 Lys Tyr AsnAsp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys Asp 610 615 620 gga tgcact ggg ccc aag gac acc atc ggc att gga gcg tgt acg acg 1920 Gly Cys ThrGly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr Thr 625 630 635 640 tgcaat ttg gcc att atc aac aat gac gcc aca gta aaa cgc tgc ctg 1968 Cys AsnLeu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys Leu 645 650 655 ctgaag gac gac aag tgc ccc gat ggg tat ttc tgg gag tat gtg cat 2016 Leu LysAsp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val His 660 665 670 ccgcaa gag cag gga tcg ctg aag cca ttg gcc ggc aga gca gtt tgc 2064 Pro GlnGlu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val Cys 675 680 685 cgaaag tgc cat ccc ctt tgc gag ctg tgc acg aac tac gga tac cat 2112 Arg LysCys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr His 690 695 700 gaacag gtg tgc tcc aag tgc acc cac tac aag cga cgg gag cag tgc 2160 Glu GlnVal Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln Cys 705 710 715 720gag acc gag tgt ccg gcc gat cac tac acg gat gag gag cag cgc gag 2208 GluThr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg Glu 725 730 735tgc ttc cag cgc cac ccg gaa tgc aat ggt tgc acg ggt ccg ggt gcc 2256 CysPhe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly Ala 740 745 750gac gat tgc aag tct tgc cgc aac ttt aag ttg ttc gac gcg aat gag 2304 AspAsp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn Glu 755 760 765acg ggt ccc tat gtg aac tcc acg atg ttc aat tgc acc tcg aag tgt 2352 ThrGly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys Cys 770 775 780ccc ttg gag atg cga cat gtg aac tat cag tac acg gcc att gga ccc 2400 ProLeu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly Pro 785 790 795800 tac tgc gca gct agt ccg ccg agg agc agc aag ata act gcc aat ctg 2448Tyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn Leu 805 810815 gat gtg aac atg atc ttc att atc act ggt gct gtt ctg gtg ccg acg 2496Asp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro Thr 820 825830 atc tgc atc ctc tgc gtg gtc aca tac att tgt cgg caa aag caa aag 2544Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln Lys 835 840845 gcc aag aag gaa aca gtc aag atg acc atg gct ctg tct ggc tgc gag 2592Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys Glu 850 855860 gat tcc gag ccg ctg cgt ccc tcg aac att gga gcc aac cta tgc aag 2640Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys Lys 865 870875 880 ttg cgc att gtc aag gac gcc gag ttg cgc aag ggc gga gtc ctt gga2688 Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu Gly 885890 895 atg gga gcc ttt gga cga gtg tac aag ggc gtt tgg gtg ccg gag ggt2736 Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu Gly 900905 910 gag aac gtc aag att cca gtg gcc att aag gag ctg ctc aag tcc aca2784 Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser Thr 915920 925 ggc gcc gag tca agc gaa gag ttc ctc cgc gaa gcc tac atc atg gcc2832 Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met Ala 930935 940 tct gag gag cac gtt aat ctg ctg aag ctc ctg gcc gtg tgc atg tcc2880 Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met Ser 945950 955 960 tca caa atg atg cta atc acg caa ctg atg ccg ctt ggc tgc ctgttg 2928 Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys Leu Leu965 970 975 gac tat gtg cga aat aac cgg gac aag atc ggc tct aag gct ctgctc 2976 Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys Ala Leu Leu980 985 990 aac tgg agc acg caa atc gcc aag ggc atg tcg tat ctg gag gagaag 3024 Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Glu Lys995 1000 1005 cga ctg gtc cac aga gac ttg gct gcc cgc aat gtc ctg gtgcag 3069 Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gln1010 1015 1020 act ccc tcg ctg gtg aag atc acc gac ttt ggg ctg gcc aagttg 3114 Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu1025 1030 1035 ctg agc agc gat tcc aat gag tac aag gct gct ggc ggc aagatg 3159 Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala Gly Gly Lys Met1040 1045 1050 ccc atc aag tgg ttg gca ctg gag tgc atc cgc aat cgt gtattc 3204 Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg Asn Arg Val Phe1055 1060 1065 acc agc aag tcc gat gtc tgg gcc ttt ggt gtg acc att tgggaa 3249 Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val Thr Ile Trp Glu1070 1075 1080 ctg ctg acc ttt ggc cag cgt cca cac gag aac atc cca gctaag 3294 Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn Ile Pro Ala Lys1085 1090 1095 gat att ccc gat ctt att gaa gtc ggt ctg aag ctg gag cagccg 3339 Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys Leu Glu Gln Pro1100 1105 1110 gag att tgt tcg ctg gac att tac tgt aca ctg ctc tcg tgctgg 3384 Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu Leu Ser Cys Trp1115 1120 1125 cac ttg gat gcc gcc atg cgt cca acc ttc aag cag ctg actacg 3429 His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys Gln Leu Thr Thr1130 1135 1140 gtc ttt gct gag ttc gcc aga gat ccg ggt cgc tat ctg gccatt 3474 Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg Tyr Leu Ala Ile1145 1150 1155 ccc ggg gat aag ttc acc cgg ctg ccg gct tac acg agt caggat 3519 Pro Gly Asp Lys Phe Thr Arg Leu Pro Ala Tyr Thr Ser Gln Asp1160 1165 1170 gag aag gat ctc atc cga aaa ttg gct ccc acc acc gat gggtcc 3564 Glu Lys Asp Leu Ile Arg Lys Leu Ala Pro Thr Thr Asp Gly Ser1175 1180 1185 gaa gcc att gcg aaa ccc gat gac tac ctg caa ccc aag gcagca 3609 Glu Ala Ile Ala Lys Pro Asp Asp Tyr Leu Gln Pro Lys Ala Ala1190 1195 1200 cct ggt cct agt cac aga acc gactgcacgg atgagatgcccaagctgaac 3660 Pro Gly Pro Ser His Arg Thr 1205 1210 cgctactgcaaggatcctag taacaagaat tcgagtaccg gagacgatga gagggattcg 3720 agtgcccgggaagtgggcgt gggtaatctg cgcctcgatc taccagtcga tgaggatgat 3780 tatctgatgcccacttgcca accgggtccc aacaacaaca acaacatgaa taatcccaat 3840 caaaacaatatggcagctgt gggcgtggct gccggctaca tggatctcat cggagtgccc 3900 gttagtgtggacaatccgga gtatctgcta aacgcgcaga cactgggagt tggggagtcg 3960 ccgatacccacccagaccat cgggataccg gtgatgggag gcccgggcac catggaggtc 4020 aaggtgccaatgccaggcag tgagccaacc agctccgatc acgagtacta caatgatacc 4080 caacgggagttgcagccact gcatcgaaac cgcaacacgg agacgagggt gtag 4134 22 1210 PRT1-O(Drosophila melanogaster) 22 Met Met Ile Ile Ser Met Trp Met Ser IleSer Arg Gly Leu Trp Asp 1 5 10 15 Ser Ser Ser Ile Leu Ser Val Leu LeuIle Leu Ala Cys Met Ala Ser 20 25 30 Ile Thr Thr Ser Ser Ser Val Ser AsnAla Gly Tyr Val Asp Asn Gly 35 40 45 Asn Met Lys Val Cys Ile Gly Thr LysSer Arg Leu Ser Val Pro Ser 50 55 60 Asn Lys Glu His His Tyr Arg Asn LeuArg Asp Arg Tyr Thr Asn Cys 65 70 75 80 Thr Tyr Val Asp Gly Asn Leu LysLeu Thr Trp Leu Pro Asn Glu Asn 85 90 95 Leu Asp Leu Ser Phe Leu Asp AsnIle Arg Glu Val Thr Gly Tyr Ile 100 105 110 Leu Ile Ser His Val Asp ValLys Lys Val Val Phe Pro Lys Leu Gln 115 120 125 Ile Ile Arg Gly Arg ThrLeu Phe Ser Leu Ser Val Glu Glu Glu Lys 130 135 140 Tyr Ala Leu Phe ValThr Tyr Ser Lys Met Tyr Thr Leu Glu Ile Pro 145 150 155 160 Asp Leu ArgAsp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn Tyr 165 170 175 Asn LeuCys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser Asn 180 185 190 GlyThr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg Glu 195 200 205Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu Gly 210 215220 Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser Pro Gln Cys 225230 235 240 Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu Cys Cys HisLeu 245 250 255 Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln Lys Asp CysIle Ala 260 265 270 Cys Lys Asn Phe Phe Asp Glu Ala Val Ser Lys Glu GluCys Pro Pro 275 280 285 Met Arg Lys Tyr Asn Pro Thr Thr Tyr Val Leu GluThr Asn Pro Glu 290 295 300 Gly Lys Tyr Ala Tyr Gly Ala Thr Cys Val LysGlu Cys Pro Gly His 305 310 315 320 Leu Leu Arg Asp Asn Gly Ala Cys ValArg Ser Cys Pro Gln Asp Lys 325 330 335 Met Asp Lys Gly Gly Glu Cys ValPro Cys Asn Gly Pro Cys Pro Lys 340 345 350 Thr Cys Pro Gly Val Thr ValLeu His Ala Gly Asn Ile Asp Ser Phe 355 360 365 Arg Asn Cys Thr Val IleAsp Gly Asn Ile Arg Ile Leu Asp Gln Thr 370 375 380 Phe Ser Gly Phe GlnAsp Val Tyr Ala Asn Tyr Thr Met Gly Pro Arg 385 390 395 400 Tyr Ile ProLeu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val Lys 405 410 415 Glu IleThr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe Arg 420 425 430 AsnLeu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln Leu 435 440 445Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu Tyr 450 455460 Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val Val 465470 475 480 Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn Ile Arg TrpPro 485 490 495 Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp Val Asn GluAsn Leu 500 505 510 Arg Ala Asp Leu Cys Glu Lys Asn Gly Thr Ile Cys SerAsp Gln Cys 515 520 525 Asn Glu Asp Gly Cys Trp Gly Ala Gly Thr Asp GlnCys Leu Thr Cys 530 535 540 Lys Asn Phe Asn Phe Asn Gly Thr Cys Ile AlaAsp Cys Gly Tyr Ile 545 550 555 560 Ser Asn Ala Tyr Lys Phe Asp Asn ArgThr Cys Lys Ile Cys His Pro 565 570 575 Glu Cys Arg Thr Cys Asn Gly AlaGly Ala Asp His Cys Gln Glu Cys 580 585 590 Val His Val Arg Asp Gly GlnHis Cys Val Ser Glu Cys Pro Lys Asn 595 600 605 Lys Tyr Asn Asp Arg GlyVal Cys Arg Glu Cys His Ala Thr Cys Asp 610 615 620 Gly Cys Thr Gly ProLys Asp Thr Ile Gly Ile Gly Ala Cys Thr Thr 625 630 635 640 Cys Asn LeuAla Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys Leu 645 650 655 Leu LysAsp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val His 660 665 670 ProGln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val Cys 675 680 685Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr His 690 695700 Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln Cys 705710 715 720 Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln ArgGlu 725 730 735 Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly ProGly Ala 740 745 750 Asp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe AspAla Asn Glu 755 760 765 Thr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn CysThr Ser Lys Cys 770 775 780 Pro Leu Glu Met Arg His Val Asn Tyr Gln TyrThr Ala Ile Gly Pro 785 790 795 800 Tyr Cys Ala Ala Ser Pro Pro Arg SerSer Lys Ile Thr Ala Asn Leu 805 810 815 Asp Val Asn Met Ile Phe Ile IleThr Gly Ala Val Leu Val Pro Thr 820 825 830 Ile Cys Ile Leu Cys Val ValThr Tyr Ile Cys Arg Gln Lys Gln Lys 835 840 845 Ala Lys Lys Glu Thr ValLys Met Thr Met Ala Leu Ser Gly Cys Glu 850 855 860 Asp Ser Glu Pro LeuArg Pro Ser Asn Ile Gly Ala Asn Leu Cys Lys 865 870 875 880 Leu Arg IleVal Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu Gly 885 890 895 Met GlyAla Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu Gly 900 905 910 GluAsn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser Thr 915 920 925Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met Ala 930 935940 Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met Ser 945950 955 960 Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys LeuLeu 965 970 975 Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys AlaLeu Leu 980 985 990 Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr LeuGlu Glu Lys 995 1000 1005 Arg Leu Val His Arg Asp Leu Ala Ala Arg AsnVal Leu Val Gln 1010 1015 1020 Thr Pro Ser Leu Val Lys Ile Thr Asp PheGly Leu Ala Lys Leu 1025 1030 1035 Leu Ser Ser Asp Ser Asn Glu Tyr LysAla Ala Gly Gly Lys Met 1040 1045 1050 Pro Ile Lys Trp Leu Ala Leu GluCys Ile Arg Asn Arg Val Phe 1055 1060 1065 Thr Ser Lys Ser Asp Val TrpAla Phe Gly Val Thr Ile Trp Glu 1070 1075 1080 Leu Leu Thr Phe Gly GlnArg Pro His Glu Asn Ile Pro Ala Lys 1085 1090 1095 Asp Ile Pro Asp LeuIle Glu Val Gly Leu Lys Leu Glu Gln Pro 1100 1105 1110 Glu Ile Cys SerLeu Asp Ile Tyr Cys Thr Leu Leu Ser Cys Trp 1115 1120 1125 His Leu AspAla Ala Met Arg Pro Thr Phe Lys Gln Leu Thr Thr 1130 1135 1140 Val PheAla Glu Phe Ala Arg Asp Pro Gly Arg Tyr Leu Ala Ile 1145 1150 1155 ProGly Asp Lys Phe Thr Arg Leu Pro Ala Tyr Thr Ser Gln Asp 1160 1165 1170Glu Lys Asp Leu Ile Arg Lys Leu Ala Pro Thr Thr Asp Gly Ser 1175 11801185 Glu Ala Ile Ala Lys Pro Asp Asp Tyr Leu Gln Pro Lys Ala Ala 11901195 1200 Pro Gly Pro Ser His Arg Thr 1205 1210 23 4281 DNA1-O(Drosophila melanogaster) 23 atgctgctgc gacggcgcaa cggcccctgccccttccccc tgctgcttct gctcctggcc 60 cactgcattt gcatttggcc cgcgtcggcggcccgcgatc gctacgcccg ccagaacaat 120 cgccagcgcc atcaggatat agatcgcgatcgggatcgag atcgattcct ataccgcagc 180 agttcggccc aaaatcgaca gaggggcggggccaacttcg ccctgggact gggagccaac 240 ggagtcacca ttcccaccag tctggaggataagaacaaga acgagttcgt caaggggaaa 300 atctgcatcg gcactaaatc tcggctctccgtgccctcca acaaggaaca tcattaccga 360 aacctcagag atcggtacac gaactgtacgtatgtggatg gcaacttgaa actgacctgg 420 ctacccaacg agaatttgga cctcagcttcctagacaaca tacgggaggt caccggctat 480 attctgatca gtcatgtgga cgttaagaaagtggtgtttc ccaaactaca aatcattcgc 540 ggacgcacgc tgttcagctt atccgtggaggaggagaagt atgccttgtt cgtcacttat 600 tccaaaatgt acacgctgga gattcccgatctacgcgatg tcttaaatgg ccaagtgggc 660 ttccacaaca actacaatct ctgccacatgcgaacgatcc agtggtcgga gattgtatcc 720 aacggcacgg atgcatacta caactacgactttactgctc cggagcgcga gtgtcccaag 780 tgccacgaga gctgcacgca cggatgttggggcgagggtc ccaagaattg ccagaagttc 840 agcaagctca cctgctcgcc acagtgtgccggaggtcgtt gctatggacc aaagccgcgg 900 gagtgttgtc acctcttctg cgccggaggatgcactggtc ccacgcaaaa ggattgcatc 960 gcctgcaaga acttcttcga cgaggcagtatcaaaggagg aatgcccgcc catgcgcaag 1020 tacaatccca ccacctatgt tcttgaaacgaatcctgagg gaaagtatgc ctatggtgcc 1080 acctgcgtca aggagtgtcc cggtcatctgttgcgggata atggcgcctg cgtgcgcagc 1140 tgtccccagg acaagatgga caaggggggcgagtgtgtgc cctgcaatgg accgtgcccc 1200 aaaacctgcc cgggcgttac tgtcctgcatgccggcaaca ttgactcgtt ccggaattgt 1260 acggtgatcg atggcaacat tcgcattttggatcagacct tctcgggctt ccaggatgtc 1320 tatgccaact acacgatggg accacgatacataccgctgg atcccgagcg acgggaggtg 1380 ttctccacgg tgaaggagat caccgggtatctgaatatcg agggaaccca cccgcagttc 1440 cggaatctgt cgtactttcg caatctggaaacaattcatg gccgccagct gatggagagc 1500 atgtttgccg ctttggcgat cgttaagtcatccctgtaca gcctggagat gcgcaatctg 1560 aagcagatta gttccggcag tgtggtcatccagcataata gagacctctg ctacgtaagc 1620 aatatccgtt ggccggccat tcagaaggagcccgaacaga aggtgtgggt caacgagaat 1680 ctcagggcgg atctatgcga gaaaaatggaaccatttgct cggatcagtg caacgaggac 1740 ggctgctggg gagctggcac ggatcagtgccttacctgca agaacttcaa tttcaatggc 1800 acctgcatcg ccgactgtgg ttatatatccaatgcctaca agtttgacaa tagaacgtgc 1860 aagatatgcc atccagagtg ccggacttgcaatggagctg gagcagatca ctgccaggag 1920 tgcgtccatg tgagggacgg tcagcactgtgtgtccgagt gcccgaagaa caagtacaac 1980 gatcgtggtg tctgccgaga gtgccacgccacctgcgatg gatgcactgg gcccaaggac 2040 accatcggca ttggagcgtg tacgacgtgcaatttggcca ttatcaacaa tgacgccaca 2100 gtaaaacgct gcctgctgaa ggacgacaagtgccccgatg ggtatttctg ggagtatgtg 2160 catccgcaag agcagggatc gctgaagccattggccggca gagcagtttg ccgaaagtgc 2220 catccccttt gcgagctgtg cacgaactacggataccatg aacaggtgtg ctccaagtgc 2280 acccactaca agcgacggga gcagtgcgagaccgagtgtc cggccgatca ctacacggat 2340 gaggagcagc gcgagtgctt ccagcgccacccggaatgca atggttgcac gggtccgggt 2400 gccgacgatt gcaagtcttg ccgcaactttaagttgttcg acgcgaatga gacgggtccc 2460 tatgtgaact ccacgatgtt caattgcacctcgaagtgtc ccttggagat gcgacatgtg 2520 aactatcagt acacggccat tggaccctactgcgcagcta gtccgccgag gagcagcaag 2580 ataactgcca atctggatgt gaacatgatcttcattatca ctggtgctgt tctggtgccg 2640 acgatctgca tcctctgcgt ggtcacatacatttgtcggc aaaagcaaaa ggccaagaag 2700 gaaacagtca agatgaccat ggctctgtctggctgcgagg attccgagcc gctgcgtccc 2760 tcgaacattg gagccaacct atgcaagttgcgcattgtca aggacgccga gttgcgcaag 2820 ggcggagtcc ttggaatggg agcctttggacgagtgtaca agggcgtttg ggtgccggag 2880 ggtgagaacg tcaagattcc agtggccattaaggagctgc tcaagtccac aggcgccgag 2940 tcaagcgaag agttcctccg cgaagcctacatcatggcct ctgaggagca cgttaatctg 3000 ctgaagctcc tggccgtgtg catgtcctcacaaatgatgc taatcacgca actgatgccg 3060 cttggctgcc tgttggacta tgtgcgaaataaccgggaca agatcggctc taaggctctg 3120 ctcaactgga gcacgcaaat cgccaagggcatgtcgtatc tggaggagaa gcgactggtc 3180 cacagagact tggctgcccg caatgtcctggtgcagactc cctcgctggt gaagatcacc 3240 gactttgggc tggccaagtt gctgagcagcgattccaatg agtacaaggc tgctggcggc 3300 aagatgccca tcaagtggtt ggcactggagtgcatccgca atcgtgtatt caccagcaag 3360 tccgatgtct gggcctttgg tgtgaccatttgggaactgc tgacctttgg ccagcgtcca 3420 cacgagaaca tcccagctaa ggatattcccgatcttattg aagtcggtct gaagctggag 3480 cagccggaga tttgttcgct ggacatttactgtacactgc tctcgtgctg gcacttggat 3540 gccgccatgc gtccaacctt caagcagctgactacggtct ttgctgagtt cgccagagat 3600 ccgggtcgct atctggccat tcccggggataagttcaccc ggctgccggc ttacacgagt 3660 caggatgaga aggatctcat ccgaaaattggctcccacca ccgatgggtc cgaagccatt 3720 gcgaaacccg atgactacct gcaacccaaggcagcacctg gtcctagtca cagaaccgac 3780 tgcacggatg agatgcccaa gctgaaccgctactgcaagg atcctagtaa caagaattcg 3840 agtaccggag acgatgagag ggattcgagtgcccgggaag tgggcgtggg taatctgcgc 3900 ctcgatctac cagtcgatga ggatgattatctgatgccca cttgccaacc gggtcccaac 3960 aacaacaaca acatgaataa tcccaatcaaaacaatatgg cagctgtggg cgtggctgcc 4020 ggctacatgg atctcatcgg agtgcccgttagtgtggaca atccggagta tctgctaaac 4080 gcgcagacac tgggagttgg ggagtcgccgatacccaccc agaccatcgg gataccggtg 4140 atgggaggcc cgggcaccat ggaggtcaaggtgccaatgc caggcagtga gccaaccagc 4200 tccgatcacg agtactacaa tgatacccaacgggagttgc agccactgca tcgaaaccgc 4260 aacacggaga cgagggtgta g 4281 244281 DNA E (Rattus norvegicus) CDS (1)..(3627) 24 atg ctg ctg cga cggcgc aac ggc ccc tgc ccc ttc ccc ctg ctg ctt 48 Met Leu Leu Arg Arg ArgAsn Gly Pro Cys Pro Phe Pro Leu Leu Leu 1 5 10 15 ctg ctc ctg gcc cactgc att tgc att tgg ccc gcg tcg gcg gcc cgc 96 Leu Leu Leu Ala His CysIle Cys Ile Trp Pro Ala Ser Ala Ala Arg 20 25 30 gat cgc tac gcc cgc cagaac aat cgc cag cgc cat cag gat ata gat 144 Asp Arg Tyr Ala Arg Gln AsnAsn Arg Gln Arg His Gln Asp Ile Asp 35 40 45 cgc gat cgg gat cga gat cgattc cta tac cgc agc agt tcg gcc caa 192 Arg Asp Arg Asp Arg Asp Arg PheLeu Tyr Arg Ser Ser Ser Ala Gln 50 55 60 aat cga cag agg ggc ggg gcc aacttc gcc ctg gga ctg gga gcc aac 240 Asn Arg Gln Arg Gly Gly Ala Asn PheAla Leu Gly Leu Gly Ala Asn 65 70 75 80 gga gtc acc att ccc acc agt ctggag gat aag aac aag aac gag ttc 288 Gly Val Thr Ile Pro Thr Ser Leu GluAsp Lys Asn Lys Asn Glu Phe 85 90 95 gtc aag ggg aaa atc tgc atc ggc actaaa tct cgg ctc tcc gtg ccc 336 Val Lys Gly Lys Ile Cys Ile Gly Thr LysSer Arg Leu Ser Val Pro 100 105 110 tcc aac aag gaa cat cat tac cga aacctc aga gat cgg tac acg aac 384 Ser Asn Lys Glu His His Tyr Arg Asn LeuArg Asp Arg Tyr Thr Asn 115 120 125 tgt acg tat gtg gat ggc aac ttg aaactg acc tgg cta ccc aac gag 432 Cys Thr Tyr Val Asp Gly Asn Leu Lys LeuThr Trp Leu Pro Asn Glu 130 135 140 aat ttg gac ctc agc ttc cta gac aacata cgg gag gtc acc ggc tat 480 Asn Leu Asp Leu Ser Phe Leu Asp Asn IleArg Glu Val Thr Gly Tyr 145 150 155 160 att ctg atc agt cat gtg gac gttaag aaa gtg gtg ttt ccc aaa cta 528 Ile Leu Ile Ser His Val Asp Val LysLys Val Val Phe Pro Lys Leu 165 170 175 caa atc att cgc gga cgc acg ctgttc agc tta tcc gtg gag gag gag 576 Gln Ile Ile Arg Gly Arg Thr Leu PheSer Leu Ser Val Glu Glu Glu 180 185 190 aag tat gcc ttg ttc gtc act tattcc aaa atg tac acg ctg gag att 624 Lys Tyr Ala Leu Phe Val Thr Tyr SerLys Met Tyr Thr Leu Glu Ile 195 200 205 ccc gat cta cgc gat gtc tta aatggc caa gtg ggc ttc cac aac aac 672 Pro Asp Leu Arg Asp Val Leu Asn GlyGln Val Gly Phe His Asn Asn 210 215 220 tac aat ctc tgc cac atg cga acgatc cag tgg tcg gag att gta tcc 720 Tyr Asn Leu Cys His Met Arg Thr IleGln Trp Ser Glu Ile Val Ser 225 230 235 240 aac ggc acg gat gca tac tacaac tac gac ttt act gct ccg gag cgc 768 Asn Gly Thr Asp Ala Tyr Tyr AsnTyr Asp Phe Thr Ala Pro Glu Arg 245 250 255 gag tgt ccc aag tgc cac gagagc tgc acg cac gga tgt tgg ggc gag 816 Glu Cys Pro Lys Cys His Glu SerCys Thr His Gly Cys Trp Gly Glu 260 265 270 ggt ccc aag aat tgc cag aagttc agc aag ctc acc tgc tcg cca cag 864 Gly Pro Lys Asn Cys Gln Lys PheSer Lys Leu Thr Cys Ser Pro Gln 275 280 285 tgt gcc gga ggt cgt tgc tatgga cca aag ccg cgg gag tgt tgt cac 912 Cys Ala Gly Gly Arg Cys Tyr GlyPro Lys Pro Arg Glu Cys Cys His 290 295 300 ctc ttc tgc gcc gga gga tgcact ggt ccc acg caa aag gat tgc atc 960 Leu Phe Cys Ala Gly Gly Cys ThrGly Pro Thr Gln Lys Asp Cys Ile 305 310 315 320 gcc tgc aag aac ttc ttcgac gag gca gta tca aag gag gaa tgc ccg 1008 Ala Cys Lys Asn Phe Phe AspGlu Ala Val Ser Lys Glu Glu Cys Pro 325 330 335 ccc atg cgc aag tac aatccc acc acc tat gtt ctt gaa acg aat cct 1056 Pro Met Arg Lys Tyr Asn ProThr Thr Tyr Val Leu Glu Thr Asn Pro 340 345 350 gag gga aag tat gcc tatggt gcc acc tgc gtc aag gag tgt ccc ggt 1104 Glu Gly Lys Tyr Ala Tyr GlyAla Thr Cys Val Lys Glu Cys Pro Gly 355 360 365 cat ctg ttg cgg gat aatggc gcc tgc gtg cgc agc tgt ccc cag gac 1152 His Leu Leu Arg Asp Asn GlyAla Cys Val Arg Ser Cys Pro Gln Asp 370 375 380 aag atg gac aag ggg ggcgag tgt gtg ccc tgc aat gga ccg tgc ccc 1200 Lys Met Asp Lys Gly Gly GluCys Val Pro Cys Asn Gly Pro Cys Pro 385 390 395 400 aaa acc tgc ccg ggcgtt act gtc ctg cat gcc ggc aac att gac tcg 1248 Lys Thr Cys Pro Gly ValThr Val Leu His Ala Gly Asn Ile Asp Ser 405 410 415 ttc cgg aat tgt acggtg atc gat ggc aac att cgc att ttg gat cag 1296 Phe Arg Asn Cys Thr ValIle Asp Gly Asn Ile Arg Ile Leu Asp Gln 420 425 430 acc ttc tcg ggc ttccag gat gtc tat gcc aac tac acg atg gga cca 1344 Thr Phe Ser Gly Phe GlnAsp Val Tyr Ala Asn Tyr Thr Met Gly Pro 435 440 445 cga tac ata ccg ctggat ccc gag cga cgg gag gtg ttc tcc acg gtg 1392 Arg Tyr Ile Pro Leu AspPro Glu Arg Arg Glu Val Phe Ser Thr Val 450 455 460 aag gag atc acc gggtat ctg aat atc gag gga acc cac ccg cag ttc 1440 Lys Glu Ile Thr Gly TyrLeu Asn Ile Glu Gly Thr His Pro Gln Phe 465 470 475 480 cgg aat ctg tcgtac ttt cgc aat ctg gaa aca att cat ggc cgc cag 1488 Arg Asn Leu Ser TyrPhe Arg Asn Leu Glu Thr Ile His Gly Arg Gln 485 490 495 ctg atg gag agcatg ttt gcc gct ttg gcg atc gtt aag tca tcc ctg 1536 Leu Met Glu Ser MetPhe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu 500 505 510 tac agc ctg gagatg cgc aat ctg aag cag att agt tcc ggc agt gtg 1584 Tyr Ser Leu Glu MetArg Asn Leu Lys Gln Ile Ser Ser Gly Ser Val 515 520 525 gtc atc cag cataat aga gac ctc tgc tac gta agc aat atc cgt tgg 1632 Val Ile Gln His AsnArg Asp Leu Cys Tyr Val Ser Asn Ile Arg Trp 530 535 540 ccg gcc att cagaag gag ccc gaa cag aag gtg tgg gtc aac gag aat 1680 Pro Ala Ile Gln LysGlu Pro Glu Gln Lys Val Trp Val Asn Glu Asn 545 550 555 560 ctc agg gcggat cta tgc gag aaa aat gga acc att tgc tcg gat cag 1728 Leu Arg Ala AspLeu Cys Glu Lys Asn Gly Thr Ile Cys Ser Asp Gln 565 570 575 tgc aac gaggac ggc tgc tgg gga gct ggc acg gat cag tgc ctt acc 1776 Cys Asn Glu AspGly Cys Trp Gly Ala Gly Thr Asp Gln Cys Leu Thr 580 585 590 tgc aag aacttc aat ttc aat ggc acc tgc atc gcc gac tgt ggt tat 1824 Cys Lys Asn PheAsn Phe Asn Gly Thr Cys Ile Ala Asp Cys Gly Tyr 595 600 605 ata tcc aatgcc tac aag ttt gac aat aga acg tgc aag ata tgc cat 1872 Ile Ser Asn AlaTyr Lys Phe Asp Asn Arg Thr Cys Lys Ile Cys His 610 615 620 cca gag tgccgg act tgc aat gga gct gga gca gat cac tgc cag gag 1920 Pro Glu Cys ArgThr Cys Asn Gly Ala Gly Ala Asp His Cys Gln Glu 625 630 635 640 tgc gtccat gtg agg gac ggt cag cac tgt gtg tcc gag tgc ccg aag 1968 Cys Val HisVal Arg Asp Gly Gln His Cys Val Ser Glu Cys Pro Lys 645 650 655 aac aagtac aac gat cgt ggt gtc tgc cga gag tgc cac gcc acc tgc 2016 Asn Lys TyrAsn Asp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys 660 665 670 gat ggatgc act ggg ccc aag gac acc atc ggc att gga gcg tgt acg 2064 Asp Gly CysThr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr 675 680 685 acg tgcaat ttg gcc att atc aac aat gac gcc aca gta aaa cgc tgc 2112 Thr Cys AsnLeu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys 690 695 700 ctg ctgaag gac gac aag tgc ccc gat ggg tat ttc tgg gag tat gtg 2160 Leu Leu LysAsp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val 705 710 715 720 catccg caa gag cag gga tcg ctg aag cca ttg gcc ggc aga gca gtt 2208 His ProGln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val 725 730 735 tgccga aag tgc cat ccc ctt tgc gag ctg tgc acg aac tac gga tac 2256 Cys ArgLys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr 740 745 750 catgaa cag gtg tgc tcc aag tgc acc cac tac aag cga cgg gag cag 2304 His GluGln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg Glu Gln 755 760 765 tgcgag acc gag tgt ccg gcc gat cac tac acg gat gag gag cag cgc 2352 Cys GluThr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu Glu Gln Arg 770 775 780 gagtgc ttc cag cgc cac ccg gaa tgc aat ggt tgc acg ggt ccg ggt 2400 Glu CysPhe Gln Arg His Pro Glu Cys Asn Gly Cys Thr Gly Pro Gly 785 790 795 800gcc gac gat tgc aag tct tgc cgc aac ttt aag ttg ttc gac gcg aat 2448 AlaAsp Asp Cys Lys Ser Cys Arg Asn Phe Lys Leu Phe Asp Ala Asn 805 810 815gag acg ggt ccc tat gtg aac tcc acg atg ttc aat tgc acc tcg aag 2496 GluThr Gly Pro Tyr Val Asn Ser Thr Met Phe Asn Cys Thr Ser Lys 820 825 830tgt ccc ttg gag atg cga cat gtg aac tat cag tac acg gcc att gga 2544 CysPro Leu Glu Met Arg His Val Asn Tyr Gln Tyr Thr Ala Ile Gly 835 840 845ccc tac tgc gca gct agt ccg ccg agg agc agc aag ata act gcc aat 2592 ProTyr Cys Ala Ala Ser Pro Pro Arg Ser Ser Lys Ile Thr Ala Asn 850 855 860ctg gat gtg aac atg atc ttc att atc act ggt gct gtt ctg gtg ccg 2640 LeuAsp Val Asn Met Ile Phe Ile Ile Thr Gly Ala Val Leu Val Pro 865 870 875880 acg atc tgc atc ctc tgc gtg gtc aca tac att tgt cgg caa aag caa 2688Thr Ile Cys Ile Leu Cys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln 885 890895 aag gcc aag aag gaa aca gtc aag atg acc atg gct ctg tct ggc tgc 2736Lys Ala Lys Lys Glu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys 900 905910 gag gat tcc gag ccg ctg cgt ccc tcg aac att gga gcc aac cta tgc 2784Glu Asp Ser Glu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys 915 920925 aag ttg cgc att gtc aag gac gcc gag ttg cgc aag ggc gga gtc ctt 2832Lys Leu Arg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu 930 935940 gga atg gga gcc ttt gga cga gtg tac aag ggc gtt tgg gtg ccg gag 2880Gly Met Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu 945 950955 960 ggt gag aac gtc aag att cca gtg gcc att aag gag ctg ctc aag tcc2928 Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser 965970 975 aca ggc gcc gag tca agc gaa gag ttc ctc cgc gaa gcc tac atc atg2976 Thr Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met 980985 990 gcc tct gag gag cac gtt aat ctg ctg aag ctc ctg gcc gtg tgc atg3024 Ala Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val Cys Met 9951000 1005 tcc tca caa atg atg cta atc acg caa ctg atg ccg ctt ggc tgc3069 Ser Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro Leu Gly Cys 10101015 1020 ctg ttg gac tat gtg cga aat aac cgg gac aag atc ggc tct aag3114 Leu Leu Asp Tyr Val Arg Asn Asn Arg Asp Lys Ile Gly Ser Lys 10251030 1035 gct ctg ctc aac tgg agc acg caa atc gcc aag ggc atg tcg tat3159 Ala Leu Leu Asn Trp Ser Thr Gln Ile Ala Lys Gly Met Ser Tyr 10401045 1050 ctg gag gag aag cga ctg gtc cac aga gac ttg gct gcc cgc aat3204 Leu Glu Glu Lys Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn 10551060 1065 gtc ctg gtg cag act ccc tcg ctg gtg aag atc acc gac ttt ggg3249 Val Leu Val Gln Thr Pro Ser Leu Val Lys Ile Thr Asp Phe Gly 10701075 1080 ctg gcc aag ttg ctg agc agc gat tcc aat gag tac aag gct gct3294 Leu Ala Lys Leu Leu Ser Ser Asp Ser Asn Glu Tyr Lys Ala Ala 10851090 1095 ggc ggc aag atg ccc atc aag tgg ttg gca ctg gag tgc atc cgc3339 Gly Gly Lys Met Pro Ile Lys Trp Leu Ala Leu Glu Cys Ile Arg 11001105 1110 aat cgt gta ttc acc agc aag tcc gat gtc tgg gcc ttt ggt gtg3384 Asn Arg Val Phe Thr Ser Lys Ser Asp Val Trp Ala Phe Gly Val 11151120 1125 acc att tgg gaa ctg ctg acc ttt ggc cag cgt cca cac gag aac3429 Thr Ile Trp Glu Leu Leu Thr Phe Gly Gln Arg Pro His Glu Asn 11301135 1140 atc cca gct aag gat att ccc gat ctt att gaa gtc ggt ctg aag3474 Ile Pro Ala Lys Asp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys 11451150 1155 ctg gag cag ccg gag att tgt tcg ctg gac att tac tgt aca ctg3519 Leu Glu Gln Pro Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu 11601165 1170 ctc tcg tgc tgg cac ttg gat gcc gcc atg cgt cca acc ttc aag3564 Leu Ser Cys Trp His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys 11751180 1185 cag ctg act acg gtc ttt gct gag ttc gcc aga gat ccg ggt cgc3609 Gln Leu Thr Thr Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg 11901195 1200 tat ctg gcc att ccc ggg gataagttca cccggctgcc ggcttacacg 3657Tyr Leu Ala Ile Pro Gly 1205 agtcaggatg agaaggatct catccgaaaa ttggctcccaccaccgatgg gtccgaagcc 3717 attgcgaaac ccgatgacta cctgcaaccc aaggcagcacctggtcctag tcacagaacc 3777 gactgcacgg atgagatgcc caagctgaac cgctactgcaaggatcctag taacaagaat 3837 tcgagtaccg gagacgatga gagggattcg agtgcccgggaagtgggcgt gggtaatctg 3897 cgcctcgatc taccagtcga tgaggatgat tatctgatgcccacttgcca accgggtccc 3957 aacaacaaca acaacatgaa taatcccaat caaaacaatatggcagctgt gggcgtggct 4017 gccggctaca tggatctcat cggagtgccc gttagtgtggacaatccgga gtatctgcta 4077 aacgcgcaga cactgggagt tggggagtcg ccgatacccacccagaccat cgggataccg 4137 gtgatgggag gcccgggcac catggaggtc aaggtgccaatgccaggcag tgagccaacc 4197 agctccgatc acgagtacta caatgatacc caacgggagttgcagccact gcatcgaaac 4257 cgcaacacgg agacgagggt gtag 4281 25 1209 PRT E(Rattus norvegicus) 25 Met Leu Leu Arg Arg Arg Asn Gly Pro Cys Pro PhePro Leu Leu Leu 1 5 10 15 Leu Leu Leu Ala His Cys Ile Cys Ile Trp ProAla Ser Ala Ala Arg 20 25 30 Asp Arg Tyr Ala Arg Gln Asn Asn Arg Gln ArgHis Gln Asp Ile Asp 35 40 45 Arg Asp Arg Asp Arg Asp Arg Phe Leu Tyr ArgSer Ser Ser Ala Gln 50 55 60 Asn Arg Gln Arg Gly Gly Ala Asn Phe Ala LeuGly Leu Gly Ala Asn 65 70 75 80 Gly Val Thr Ile Pro Thr Ser Leu Glu AspLys Asn Lys Asn Glu Phe 85 90 95 Val Lys Gly Lys Ile Cys Ile Gly Thr LysSer Arg Leu Ser Val Pro 100 105 110 Ser Asn Lys Glu His His Tyr Arg AsnLeu Arg Asp Arg Tyr Thr Asn 115 120 125 Cys Thr Tyr Val Asp Gly Asn LeuLys Leu Thr Trp Leu Pro Asn Glu 130 135 140 Asn Leu Asp Leu Ser Phe LeuAsp Asn Ile Arg Glu Val Thr Gly Tyr 145 150 155 160 Ile Leu Ile Ser HisVal Asp Val Lys Lys Val Val Phe Pro Lys Leu 165 170 175 Gln Ile Ile ArgGly Arg Thr Leu Phe Ser Leu Ser Val Glu Glu Glu 180 185 190 Lys Tyr AlaLeu Phe Val Thr Tyr Ser Lys Met Tyr Thr Leu Glu Ile 195 200 205 Pro AspLeu Arg Asp Val Leu Asn Gly Gln Val Gly Phe His Asn Asn 210 215 220 TyrAsn Leu Cys His Met Arg Thr Ile Gln Trp Ser Glu Ile Val Ser 225 230 235240 Asn Gly Thr Asp Ala Tyr Tyr Asn Tyr Asp Phe Thr Ala Pro Glu Arg 245250 255 Glu Cys Pro Lys Cys His Glu Ser Cys Thr His Gly Cys Trp Gly Glu260 265 270 Gly Pro Lys Asn Cys Gln Lys Phe Ser Lys Leu Thr Cys Ser ProGln 275 280 285 Cys Ala Gly Gly Arg Cys Tyr Gly Pro Lys Pro Arg Glu CysCys His 290 295 300 Leu Phe Cys Ala Gly Gly Cys Thr Gly Pro Thr Gln LysAsp Cys Ile 305 310 315 320 Ala Cys Lys Asn Phe Phe Asp Glu Ala Val SerLys Glu Glu Cys Pro 325 330 335 Pro Met Arg Lys Tyr Asn Pro Thr Thr TyrVal Leu Glu Thr Asn Pro 340 345 350 Glu Gly Lys Tyr Ala Tyr Gly Ala ThrCys Val Lys Glu Cys Pro Gly 355 360 365 His Leu Leu Arg Asp Asn Gly AlaCys Val Arg Ser Cys Pro Gln Asp 370 375 380 Lys Met Asp Lys Gly Gly GluCys Val Pro Cys Asn Gly Pro Cys Pro 385 390 395 400 Lys Thr Cys Pro GlyVal Thr Val Leu His Ala Gly Asn Ile Asp Ser 405 410 415 Phe Arg Asn CysThr Val Ile Asp Gly Asn Ile Arg Ile Leu Asp Gln 420 425 430 Thr Phe SerGly Phe Gln Asp Val Tyr Ala Asn Tyr Thr Met Gly Pro 435 440 445 Arg TyrIle Pro Leu Asp Pro Glu Arg Arg Glu Val Phe Ser Thr Val 450 455 460 LysGlu Ile Thr Gly Tyr Leu Asn Ile Glu Gly Thr His Pro Gln Phe 465 470 475480 Arg Asn Leu Ser Tyr Phe Arg Asn Leu Glu Thr Ile His Gly Arg Gln 485490 495 Leu Met Glu Ser Met Phe Ala Ala Leu Ala Ile Val Lys Ser Ser Leu500 505 510 Tyr Ser Leu Glu Met Arg Asn Leu Lys Gln Ile Ser Ser Gly SerVal 515 520 525 Val Ile Gln His Asn Arg Asp Leu Cys Tyr Val Ser Asn IleArg Trp 530 535 540 Pro Ala Ile Gln Lys Glu Pro Glu Gln Lys Val Trp ValAsn Glu Asn 545 550 555 560 Leu Arg Ala Asp Leu Cys Glu Lys Asn Gly ThrIle Cys Ser Asp Gln 565 570 575 Cys Asn Glu Asp Gly Cys Trp Gly Ala GlyThr Asp Gln Cys Leu Thr 580 585 590 Cys Lys Asn Phe Asn Phe Asn Gly ThrCys Ile Ala Asp Cys Gly Tyr 595 600 605 Ile Ser Asn Ala Tyr Lys Phe AspAsn Arg Thr Cys Lys Ile Cys His 610 615 620 Pro Glu Cys Arg Thr Cys AsnGly Ala Gly Ala Asp His Cys Gln Glu 625 630 635 640 Cys Val His Val ArgAsp Gly Gln His Cys Val Ser Glu Cys Pro Lys 645 650 655 Asn Lys Tyr AsnAsp Arg Gly Val Cys Arg Glu Cys His Ala Thr Cys 660 665 670 Asp Gly CysThr Gly Pro Lys Asp Thr Ile Gly Ile Gly Ala Cys Thr 675 680 685 Thr CysAsn Leu Ala Ile Ile Asn Asn Asp Ala Thr Val Lys Arg Cys 690 695 700 LeuLeu Lys Asp Asp Lys Cys Pro Asp Gly Tyr Phe Trp Glu Tyr Val 705 710 715720 His Pro Gln Glu Gln Gly Ser Leu Lys Pro Leu Ala Gly Arg Ala Val 725730 735 Cys Arg Lys Cys His Pro Leu Cys Glu Leu Cys Thr Asn Tyr Gly Tyr740 745 750 His Glu Gln Val Cys Ser Lys Cys Thr His Tyr Lys Arg Arg GluGln 755 760 765 Cys Glu Thr Glu Cys Pro Ala Asp His Tyr Thr Asp Glu GluGln Arg 770 775 780 Glu Cys Phe Gln Arg His Pro Glu Cys Asn Gly Cys ThrGly Pro Gly 785 790 795 800 Ala Asp Asp Cys Lys Ser Cys Arg Asn Phe LysLeu Phe Asp Ala Asn 805 810 815 Glu Thr Gly Pro Tyr Val Asn Ser Thr MetPhe Asn Cys Thr Ser Lys 820 825 830 Cys Pro Leu Glu Met Arg His Val AsnTyr Gln Tyr Thr Ala Ile Gly 835 840 845 Pro Tyr Cys Ala Ala Ser Pro ProArg Ser Ser Lys Ile Thr Ala Asn 850 855 860 Leu Asp Val Asn Met Ile PheIle Ile Thr Gly Ala Val Leu Val Pro 865 870 875 880 Thr Ile Cys Ile LeuCys Val Val Thr Tyr Ile Cys Arg Gln Lys Gln 885 890 895 Lys Ala Lys LysGlu Thr Val Lys Met Thr Met Ala Leu Ser Gly Cys 900 905 910 Glu Asp SerGlu Pro Leu Arg Pro Ser Asn Ile Gly Ala Asn Leu Cys 915 920 925 Lys LeuArg Ile Val Lys Asp Ala Glu Leu Arg Lys Gly Gly Val Leu 930 935 940 GlyMet Gly Ala Phe Gly Arg Val Tyr Lys Gly Val Trp Val Pro Glu 945 950 955960 Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Glu Leu Leu Lys Ser 965970 975 Thr Gly Ala Glu Ser Ser Glu Glu Phe Leu Arg Glu Ala Tyr Ile Met980 985 990 Ala Ser Glu Glu His Val Asn Leu Leu Lys Leu Leu Ala Val CysMet 995 1000 1005 Ser Ser Gln Met Met Leu Ile Thr Gln Leu Met Pro LeuGly Cys 1010 1015 1020 Leu Leu Asp Tyr Val Arg Asn Asn Arg Asp Lys IleGly Ser Lys 1025 1030 1035 Ala Leu Leu Asn Trp Ser Thr Gln Ile Ala LysGly Met Ser Tyr 1040 1045 1050 Leu Glu Glu Lys Arg Leu Val His Arg AspLeu Ala Ala Arg Asn 1055 1060 1065 Val Leu Val Gln Thr Pro Ser Leu ValLys Ile Thr Asp Phe Gly 1070 1075 1080 Leu Ala Lys Leu Leu Ser Ser AspSer Asn Glu Tyr Lys Ala Ala 1085 1090 1095 Gly Gly Lys Met Pro Ile LysTrp Leu Ala Leu Glu Cys Ile Arg 1100 1105 1110 Asn Arg Val Phe Thr SerLys Ser Asp Val Trp Ala Phe Gly Val 1115 1120 1125 Thr Ile Trp Glu LeuLeu Thr Phe Gly Gln Arg Pro His Glu Asn 1130 1135 1140 Ile Pro Ala LysAsp Ile Pro Asp Leu Ile Glu Val Gly Leu Lys 1145 1150 1155 Leu Glu GlnPro Glu Ile Cys Ser Leu Asp Ile Tyr Cys Thr Leu 1160 1165 1170 Leu SerCys Trp His Leu Asp Ala Ala Met Arg Pro Thr Phe Lys 1175 1180 1185 GlnLeu Thr Thr Val Phe Ala Glu Phe Ala Arg Asp Pro Gly Arg 1190 1195 1200Tyr Leu Ala Ile Pro Gly 1205 26 3576 DNA aA-Oa(Danio rerio) CDS(1)..(3573) 26 atg gca gga cca act gaa atc gga ttg ttt ttt acc tta ttgctc tcc 48 Met Ala Gly Pro Thr Glu Ile Gly Leu Phe Phe Thr Leu Leu LeuSer 1 5 10 15 ggg agc ttc tgc gcg acg ccg gaa aag aaa gtg tgt cag ggagca aac 96 Gly Ser Phe Cys Ala Thr Pro Glu Lys Lys Val Cys Gln Gly AlaAsn 20 25 30 aac aaa ctg act ctt ctg gga acg gtg gaa gac cat tat cag gttctg 144 Asn Lys Leu Thr Leu Leu Gly Thr Val Glu Asp His Tyr Gln Val Leu35 40 45 ctc aga atg tac aga aac tgc act gtg gtt ctg gag aac ctg gaa att192 Leu Arg Met Tyr Arg Asn Cys Thr Val Val Leu Glu Asn Leu Glu Ile 5055 60 aca cat ata aca gag aaa tat gac ctg tcc ttc ctc aag agc atc cag240 Thr His Ile Thr Glu Lys Tyr Asp Leu Ser Phe Leu Lys Ser Ile Gln 6570 75 80 gaa gtt ggt ggc tat gtt ctt atc gcg gtc aat acg gtt tcc aaa atc288 Glu Val Gly Gly Tyr Val Leu Ile Ala Val Asn Thr Val Ser Lys Ile 8590 95 cct ctg gag aac ctg cgc atc att cgc gga cac tca ctt tat gaa gac336 Pro Leu Glu Asn Leu Arg Ile Ile Arg Gly His Ser Leu Tyr Glu Asp 100105 110 aaa ttt gcc ttg gcc gtc ctg gtg aac ttc aac aac agc atc gaa caa384 Lys Phe Ala Leu Ala Val Leu Val Asn Phe Asn Asn Ser Ile Glu Gln 115120 125 ggc gta aaa gag ttg ccg ctg act agt tta act gaa ata ctt aag ggt432 Gly Val Lys Glu Leu Pro Leu Thr Ser Leu Thr Glu Ile Leu Lys Gly 130135 140 gga gtc aag ttt tgc agg aac gat tat tta tgt aat gtg ggg acc atc480 Gly Val Lys Phe Cys Arg Asn Asp Tyr Leu Cys Asn Val Gly Thr Ile 145150 155 160 gag tgg gcc gac atc ctg aac atg aag agc ctg cct aca atc gtgagc 528 Glu Trp Ala Asp Ile Leu Asn Met Lys Ser Leu Pro Thr Ile Val Ser165 170 175 cat aat ata agc tat gga aaa aac tgt gga aag tgc gat cca agctgt 576 His Asn Ile Ser Tyr Gly Lys Asn Cys Gly Lys Cys Asp Pro Ser Cys180 185 190 ttc aat ggc tcc tgc tgg ggc acc gga ccc gac aag tgc cag agaatg 624 Phe Asn Gly Ser Cys Trp Gly Thr Gly Pro Asp Lys Cys Gln Arg Met195 200 205 acg aaa gtg atc tgt gcg gag cag tgt tca ggg agg tgt aaa ggaccc 672 Thr Lys Val Ile Cys Ala Glu Gln Cys Ser Gly Arg Cys Lys Gly Pro210 215 220 aga ccc att gac tgc tgt aat gaa cac tgt gct gct gga tgc actgga 720 Arg Pro Ile Asp Cys Cys Asn Glu His Cys Ala Ala Gly Cys Thr Gly225 230 235 240 ccc aga cct aca gac tgt ctg gcc tgt aag gac ttc cag gatgaa ggg 768 Pro Arg Pro Thr Asp Cys Leu Ala Cys Lys Asp Phe Gln Asp GluGly 245 250 255 aca tgt aag gac gca tgt ccg cgg ctc atg ctc tac gac ccaaac aca 816 Thr Cys Lys Asp Ala Cys Pro Arg Leu Met Leu Tyr Asp Pro AsnThr 260 265 270 cac cag ctc gcg cca aac cca tat ggg aag tac agc ttt ggggca acg 864 His Gln Leu Ala Pro Asn Pro Tyr Gly Lys Tyr Ser Phe Gly AlaThr 275 280 285 tgc atc aag aca tgc cca cac aac tat gtg gtg acg gat cacggg gcc 912 Cys Ile Lys Thr Cys Pro His Asn Tyr Val Val Thr Asp His GlyAla 290 295 300 tgt gtg aga aca tgc agc cct ggc acc tat gaa gtg gat gagggt gga 960 Cys Val Arg Thr Cys Ser Pro Gly Thr Tyr Glu Val Asp Glu GlyGly 305 310 315 320 gtt cgc aaa tgt aag agg tgt gaa ggc ctg tgt cca aaagtg tgc aat 1008 Val Arg Lys Cys Lys Arg Cys Glu Gly Leu Cys Pro Lys ValCys Asn 325 330 335 ggg ttg gga atg ggg cct tta gcc aat gtc ctg tca atcaat gcc acc 1056 Gly Leu Gly Met Gly Pro Leu Ala Asn Val Leu Ser Ile AsnAla Thr 340 345 350 aac atc gac tcc ttt gag aac tgc act aaa atc agc ggcaat gtt gcc 1104 Asn Ile Asp Ser Phe Glu Asn Cys Thr Lys Ile Ser Gly AsnVal Ala 355 360 365 atc ctc agc acc aca ttc aga ggt gac cca cat act aacact tca gga 1152 Ile Leu Ser Thr Thr Phe Arg Gly Asp Pro His Thr Asn ThrSer Gly 370 375 380 ctg gat cca gca aag ctc agt gta ttg agt act gtc aaagaa atc act 1200 Leu Asp Pro Ala Lys Leu Ser Val Leu Ser Thr Val Lys GluIle Thr 385 390 395 400 ggt tac ctg atg att cag ctg tgg ccg gag agc atgcag tcc ctt agt 1248 Gly Tyr Leu Met Ile Gln Leu Trp Pro Glu Ser Met GlnSer Leu Ser 405 410 415 gcc ttc gaa aac ctt gag gtc atc cga gga cgg acaaaa aca caa gga 1296 Ala Phe Glu Asn Leu Glu Val Ile Arg Gly Arg Thr LysThr Gln Gly 420 425 430 acg tac agc ttt gct gtc acc aag acg gcc atc actcat tta ggc atg 1344 Thr Tyr Ser Phe Ala Val Thr Lys Thr Ala Ile Thr HisLeu Gly Met 435 440 445 cgt tct ctg agg gag atc agt gac ggg gac gtg tccatc gtt aag aat 1392 Arg Ser Leu Arg Glu Ile Ser Asp Gly Asp Val Ser IleVal Lys Asn 450 455 460 aag aat ctc tgc tac agc agc cct gaa cac tgg aaacgc ctc ttc aag 1440 Lys Asn Leu Cys Tyr Ser Ser Pro Glu His Trp Lys ArgLeu Phe Lys 465 470 475 480 tcc aaa caa cag tcg gtc aaa atg att gaa aatatg gat gct gcc acc 1488 Ser Lys Gln Gln Ser Val Lys Met Ile Glu Asn MetAsp Ala Ala Thr 485 490 495 tgc gcc aat cag aac agc aca tgt aat gag atgtgc acg gct gac ggc 1536 Cys Ala Asn Gln Asn Ser Thr Cys Asn Glu Met CysThr Ala Asp Gly 500 505 510 tgc tgg ggt ccc ggc ccc acc atg tgc ttc ggctgt gag cat tac agc 1584 Cys Trp Gly Pro Gly Pro Thr Met Cys Phe Gly CysGlu His Tyr Ser 515 520 525 cgc gga aaa cac tgc gtg gct tcc tgc aac ctgctg aat ggt gag ccg 1632 Arg Gly Lys His Cys Val Ala Ser Cys Asn Leu LeuAsn Gly Glu Pro 530 535 540 cgt gaa tat gag gtc aat aaa aca tgc atg gaatgc gat cct gaa tgt 1680 Arg Glu Tyr Glu Val Asn Lys Thr Cys Met Glu CysAsp Pro Glu Cys 545 550 555 560 ctg ctc atg aat gaa acc cag acc tgc aacggc cct gga ccc gac aaa 1728 Leu Leu Met Asn Glu Thr Gln Thr Cys Asn GlyPro Gly Pro Asp Lys 565 570 575 tgt aca gtg tgt gca aac tat aaa gac ggaccg cac tgt gtg cat cgc 1776 Cys Thr Val Cys Ala Asn Tyr Lys Asp Gly ProHis Cys Val His Arg 580 585 590 tgc ccg caa ggt gta cca gga gag aaa gacaca ctc atc tgg aaa tac 1824 Cys Pro Gln Gly Val Pro Gly Glu Lys Asp ThrLeu Ile Trp Lys Tyr 595 600 605 gct gac gtg aca cac gtt tgc cag ccc tgtcat gaa aac tgc acc cag 1872 Ala Asp Val Thr His Val Cys Gln Pro Cys HisGlu Asn Cys Thr Gln 610 615 620 gga tgt acg ggg cct gat cta aag gac tgcaaa gat ttc aaa agc tct 1920 Gly Cys Thr Gly Pro Asp Leu Lys Asp Cys LysAsp Phe Lys Ser Ser 625 630 635 640 ggt ttg ccg atg atc gct gct ggc gttgtc gga ggt cta ctg gcg ttt 1968 Gly Leu Pro Met Ile Ala Ala Gly Val ValGly Gly Leu Leu Ala Phe 645 650 655 gtt att ctg gct ctt gga gtg gcc gttctc ctg cgc aga cgc cac atc 2016 Val Ile Leu Ala Leu Gly Val Ala Val LeuLeu Arg Arg Arg His Ile 660 665 670 cgg agg aag agg act ctg aga cga ctcctg caa gag aga gag ctt gtg 2064 Arg Arg Lys Arg Thr Leu Arg Arg Leu LeuGln Glu Arg Glu Leu Val 675 680 685 gag cct ctg acc ccc agc ggc gaa gccccc aat cag gcc tta ctg cgc 2112 Glu Pro Leu Thr Pro Ser Gly Glu Ala ProAsn Gln Ala Leu Leu Arg 690 695 700 atc ctc aaa gag acg gag ttc aag aagatc aaa gtg ctg ggc tcc ggg 2160 Ile Leu Lys Glu Thr Glu Phe Lys Lys IleLys Val Leu Gly Ser Gly 705 710 715 720 gct ttc ggc act gtg cac aag ggcctt tgg gtt cca gaa gga gag aat 2208 Ala Phe Gly Thr Val His Lys Gly LeuTrp Val Pro Glu Gly Glu Asn 725 730 735 gtg aag atc cct gtc gcc atc aaggtc tta aga gaa gcc acg tct ccc 2256 Val Lys Ile Pro Val Ala Ile Lys ValLeu Arg Glu Ala Thr Ser Pro 740 745 750 aaa gct aac aag gag ata atg gatgag gcg tac gtc atg gcc agt gtt 2304 Lys Ala Asn Lys Glu Ile Met Asp GluAla Tyr Val Met Ala Ser Val 755 760 765 gag cat ccc cat gtg tgt cgt ctgctg ggc atc tgc ttg acc tcc aca 2352 Glu His Pro His Val Cys Arg Leu LeuGly Ile Cys Leu Thr Ser Thr 770 775 780 gtg cag ctc atc act cag ctg atgccc tac ggc tgc ttg ctg gac tat 2400 Val Gln Leu Ile Thr Gln Leu Met ProTyr Gly Cys Leu Leu Asp Tyr 785 790 795 800 gtc aga gaa aac aag gac cgcatc ggc tcc cag cac ctg ctc aac tgg 2448 Val Arg Glu Asn Lys Asp Arg IleGly Ser Gln His Leu Leu Asn Trp 805 810 815 tgc gtg cag atc gct aaa ggtatg aat tat cta gaa gag cgc cat ctt 2496 Cys Val Gln Ile Ala Lys Gly MetAsn Tyr Leu Glu Glu Arg His Leu 820 825 830 gtg cac cga gac ctg gca gcacgt aat gtg ttg gta aag acg cct cag 2544 Val His Arg Asp Leu Ala Ala ArgAsn Val Leu Val Lys Thr Pro Gln 835 840 845 cat gtc aag att acc gat ttcggc ctc gcc aag ctg tta aac gcg gac 2592 His Val Lys Ile Thr Asp Phe GlyLeu Ala Lys Leu Leu Asn Ala Asp 850 855 860 gag aag gag tat cac gca gatgga gga aag gtt cca att aaa tgg atg 2640 Glu Lys Glu Tyr His Ala Asp GlyGly Lys Val Pro Ile Lys Trp Met 865 870 875 880 gcg ctg gag tcc atc cagcac agg act tac acc cac cag agt gac gtc 2688 Ala Leu Glu Ser Ile Gln HisArg Thr Tyr Thr His Gln Ser Asp Val 885 890 895 tgg agc tac ggt gtg accgtc tgg gag ttg atg acg ttt ggg acg aaa 2736 Trp Ser Tyr Gly Val Thr ValTrp Glu Leu Met Thr Phe Gly Thr Lys 900 905 910 cct tat gat ggg att cctgcc agt gaa atc gcc gga gtt ctg gaa aaa 2784 Pro Tyr Asp Gly Ile Pro AlaSer Glu Ile Ala Gly Val Leu Glu Lys 915 920 925 gga gaa aga ctt cct caaccc ccc atc tgc acc att gat gtg tac atg 2832 Gly Glu Arg Leu Pro Gln ProPro Ile Cys Thr Ile Asp Val Tyr Met 930 935 940 atc atg gtc aaa tgt tggatg att gat gct gag agc aga ccc cgc ttc 2880 Ile Met Val Lys Cys Trp MetIle Asp Ala Glu Ser Arg Pro Arg Phe 945 950 955 960 agg gag ctc atc gcagag ttc act aaa atg gct cgt gac ccg tcc cgc 2928 Arg Glu Leu Ile Ala GluPhe Thr Lys Met Ala Arg Asp Pro Ser Arg 965 970 975 tat ctg gtc att cagggg gac gac cgc atg cat tta ccc agt cct tct 2976 Tyr Leu Val Ile Gln GlyAsp Asp Arg Met His Leu Pro Ser Pro Ser 980 985 990 gac tcc aag ttc taccgc agc ctg atg agc gga gag ctg gac gag gcc 3024 Asp Ser Lys Phe Tyr ArgSer Leu Met Ser Gly Glu Leu Asp Glu Ala 995 1000 1005 gtg gac gca gacgag tat tta gtg ccc aat cac agc ttc ttc agc 3069 Val Asp Ala Asp Glu TyrLeu Val Pro Asn His Ser Phe Phe Ser 1010 1015 1020 agc ccg agc acg tcccgc aca caa ctg ctg cac tct gtg agc ctg 3114 Ser Pro Ser Thr Ser Arg ThrGln Leu Leu His Ser Val Ser Leu 1025 1030 1035 aac agc agc ttt gga aactgt aat agt aga aac ggg aat ggt tat 3159 Asn Ser Ser Phe Gly Asn Cys AsnSer Arg Asn Gly Asn Gly Tyr 1040 1045 1050 cca gtg agg gag aac agc atggtc ctg cgc tac atc cca gac ccc 3204 Pro Val Arg Glu Asn Ser Met Val LeuArg Tyr Ile Pro Asp Pro 1055 1060 1065 aca gag cgc ttt cag gag gga gacttt cag cct gcg ccg ggt tat 3249 Thr Glu Arg Phe Gln Glu Gly Asp Phe GlnPro Ala Pro Gly Tyr 1070 1075 1080 aac gaa tat atg aac cag aat gag tccagc atg atc aac cca gtg 3294 Asn Glu Tyr Met Asn Gln Asn Glu Ser Ser MetIle Asn Pro Val 1085 1090 1095 tac cag cag ccc cac gga ccc ccg cgg accctc ctc cac tcc tcc 3339 Tyr Gln Gln Pro His Gly Pro Pro Arg Thr Leu LeuHis Ser Ser 1100 1105 1110 cca gcg ctg gac gag acg gaa gag gag tat ctgaac tgc ttc aag 3384 Pro Ala Leu Asp Glu Thr Glu Glu Glu Tyr Leu Asn CysPhe Lys 1115 1120 1125 agc ccg gct ccg gct tca gtg gtg gag tat ctg aacacg tcc cac 3429 Ser Pro Ala Pro Ala Ser Val Val Glu Tyr Leu Asn Thr SerHis 1130 1135 1140 aca cag ctg ctc tcc aca aag ccc ttc ttc agc atg gacaac ccc 3474 Thr Gln Leu Leu Ser Thr Lys Pro Phe Phe Ser Met Asp Asn Pro1145 1150 1155 gac tac cag cag gac ttc tgc ccg ctg gag ctc aaa aca cacacc 3519 Asp Tyr Gln Gln Asp Phe Cys Pro Leu Glu Leu Lys Thr His Thr1160 1165 1170 aac ggg cac ctg ccg gcc gcg cag aac cag gag tac atg ggcctg 3564 Asn Gly His Leu Pro Ala Ala Gln Asn Gln Glu Tyr Met Gly Leu1175 1180 1185 gag gtg cac tag 3576 Glu Val His 1190 27 1191 PRTaA-Oa(Danio rerio) 27 Met Ala Gly Pro Thr Glu Ile Gly Leu Phe Phe ThrLeu Leu Leu Ser 1 5 10 15 Gly Ser Phe Cys Ala Thr Pro Glu Lys Lys ValCys Gln Gly Ala Asn 20 25 30 Asn Lys Leu Thr Leu Leu Gly Thr Val Glu AspHis Tyr Gln Val Leu 35 40 45 Leu Arg Met Tyr Arg Asn Cys Thr Val Val LeuGlu Asn Leu Glu Ile 50 55 60 Thr His Ile Thr Glu Lys Tyr Asp Leu Ser PheLeu Lys Ser Ile Gln 65 70 75 80 Glu Val Gly Gly Tyr Val Leu Ile Ala ValAsn Thr Val Ser Lys Ile 85 90 95 Pro Leu Glu Asn Leu Arg Ile Ile Arg GlyHis Ser Leu Tyr Glu Asp 100 105 110 Lys Phe Ala Leu Ala Val Leu Val AsnPhe Asn Asn Ser Ile Glu Gln 115 120 125 Gly Val Lys Glu Leu Pro Leu ThrSer Leu Thr Glu Ile Leu Lys Gly 130 135 140 Gly Val Lys Phe Cys Arg AsnAsp Tyr Leu Cys Asn Val Gly Thr Ile 145 150 155 160 Glu Trp Ala Asp IleLeu Asn Met Lys Ser Leu Pro Thr Ile Val Ser 165 170 175 His Asn Ile SerTyr Gly Lys Asn Cys Gly Lys Cys Asp Pro Ser Cys 180 185 190 Phe Asn GlySer Cys Trp Gly Thr Gly Pro Asp Lys Cys Gln Arg Met 195 200 205 Thr LysVal Ile Cys Ala Glu Gln Cys Ser Gly Arg Cys Lys Gly Pro 210 215 220 ArgPro Ile Asp Cys Cys Asn Glu His Cys Ala Ala Gly Cys Thr Gly 225 230 235240 Pro Arg Pro Thr Asp Cys Leu Ala Cys Lys Asp Phe Gln Asp Glu Gly 245250 255 Thr Cys Lys Asp Ala Cys Pro Arg Leu Met Leu Tyr Asp Pro Asn Thr260 265 270 His Gln Leu Ala Pro Asn Pro Tyr Gly Lys Tyr Ser Phe Gly AlaThr 275 280 285 Cys Ile Lys Thr Cys Pro His Asn Tyr Val Val Thr Asp HisGly Ala 290 295 300 Cys Val Arg Thr Cys Ser Pro Gly Thr Tyr Glu Val AspGlu Gly Gly 305 310 315 320 Val Arg Lys Cys Lys Arg Cys Glu Gly Leu CysPro Lys Val Cys Asn 325 330 335 Gly Leu Gly Met Gly Pro Leu Ala Asn ValLeu Ser Ile Asn Ala Thr 340 345 350 Asn Ile Asp Ser Phe Glu Asn Cys ThrLys Ile Ser Gly Asn Val Ala 355 360 365 Ile Leu Ser Thr Thr Phe Arg GlyAsp Pro His Thr Asn Thr Ser Gly 370 375 380 Leu Asp Pro Ala Lys Leu SerVal Leu Ser Thr Val Lys Glu Ile Thr 385 390 395 400 Gly Tyr Leu Met IleGln Leu Trp Pro Glu Ser Met Gln Ser Leu Ser 405 410 415 Ala Phe Glu AsnLeu Glu Val Ile Arg Gly Arg Thr Lys Thr Gln Gly 420 425 430 Thr Tyr SerPhe Ala Val Thr Lys Thr Ala Ile Thr His Leu Gly Met 435 440 445 Arg SerLeu Arg Glu Ile Ser Asp Gly Asp Val Ser Ile Val Lys Asn 450 455 460 LysAsn Leu Cys Tyr Ser Ser Pro Glu His Trp Lys Arg Leu Phe Lys 465 470 475480 Ser Lys Gln Gln Ser Val Lys Met Ile Glu Asn Met Asp Ala Ala Thr 485490 495 Cys Ala Asn Gln Asn Ser Thr Cys Asn Glu Met Cys Thr Ala Asp Gly500 505 510 Cys Trp Gly Pro Gly Pro Thr Met Cys Phe Gly Cys Glu His TyrSer 515 520 525 Arg Gly Lys His Cys Val Ala Ser Cys Asn Leu Leu Asn GlyGlu Pro 530 535 540 Arg Glu Tyr Glu Val Asn Lys Thr Cys Met Glu Cys AspPro Glu Cys 545 550 555 560 Leu Leu Met Asn Glu Thr Gln Thr Cys Asn GlyPro Gly Pro Asp Lys 565 570 575 Cys Thr Val Cys Ala Asn Tyr Lys Asp GlyPro His Cys Val His Arg 580 585 590 Cys Pro Gln Gly Val Pro Gly Glu LysAsp Thr Leu Ile Trp Lys Tyr 595 600 605 Ala Asp Val Thr His Val Cys GlnPro Cys His Glu Asn Cys Thr Gln 610 615 620 Gly Cys Thr Gly Pro Asp LeuLys Asp Cys Lys Asp Phe Lys Ser Ser 625 630 635 640 Gly Leu Pro Met IleAla Ala Gly Val Val Gly Gly Leu Leu Ala Phe 645 650 655 Val Ile Leu AlaLeu Gly Val Ala Val Leu Leu Arg Arg Arg His Ile 660 665 670 Arg Arg LysArg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu Val 675 680 685 Glu ProLeu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu Arg 690 695 700 IleLeu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser Gly 705 710 715720 Ala Phe Gly Thr Val His Lys Gly Leu Trp Val Pro Glu Gly Glu Asn 725730 735 Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Ala Thr Ser Pro740 745 750 Lys Ala Asn Lys Glu Ile Met Asp Glu Ala Tyr Val Met Ala SerVal 755 760 765 Glu His Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu ThrSer Thr 770 775 780 Val Gln Leu Ile Thr Gln Leu Met Pro Tyr Gly Cys LeuLeu Asp Tyr 785 790 795 800 Val Arg Glu Asn Lys Asp Arg Ile Gly Ser GlnHis Leu Leu Asn Trp 805 810 815 Cys Val Gln Ile Ala Lys Gly Met Asn TyrLeu Glu Glu Arg His Leu 820 825 830 Val His Arg Asp Leu Ala Ala Arg AsnVal Leu Val Lys Thr Pro Gln 835 840 845 His Val Lys Ile Thr Asp Phe GlyLeu Ala Lys Leu Leu Asn Ala Asp 850 855 860 Glu Lys Glu Tyr His Ala AspGly Gly Lys Val Pro Ile Lys Trp Met 865 870 875 880 Ala Leu Glu Ser IleGln His Arg Thr Tyr Thr His Gln Ser Asp Val 885 890 895 Trp Ser Tyr GlyVal Thr Val Trp Glu Leu Met Thr Phe Gly Thr Lys 900 905 910 Pro Tyr AspGly Ile Pro Ala Ser Glu Ile Ala Gly Val Leu Glu Lys 915 920 925 Gly GluArg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met 930 935 940 IleMet Val Lys Cys Trp Met Ile Asp Ala Glu Ser Arg Pro Arg Phe 945 950 955960 Arg Glu Leu Ile Ala Glu Phe Thr Lys Met Ala Arg Asp Pro Ser Arg 965970 975 Tyr Leu Val Ile Gln Gly Asp Asp Arg Met His Leu Pro Ser Pro Ser980 985 990 Asp Ser Lys Phe Tyr Arg Ser Leu Met Ser Gly Glu Leu Asp GluAla 995 1000 1005 Val Asp Ala Asp Glu Tyr Leu Val Pro Asn His Ser PhePhe Ser 1010 1015 1020 Ser Pro Ser Thr Ser Arg Thr Gln Leu Leu His SerVal Ser Leu 1025 1030 1035 Asn Ser Ser Phe Gly Asn Cys Asn Ser Arg AsnGly Asn Gly Tyr 1040 1045 1050 Pro Val Arg Glu Asn Ser Met Val Leu ArgTyr Ile Pro Asp Pro 1055 1060 1065 Thr Glu Arg Phe Gln Glu Gly Asp PheGln Pro Ala Pro Gly Tyr 1070 1075 1080 Asn Glu Tyr Met Asn Gln Asn GluSer Ser Met Ile Asn Pro Val 1085 1090 1095 Tyr Gln Gln Pro His Gly ProPro Arg Thr Leu Leu His Ser Ser 1100 1105 1110 Pro Ala Leu Asp Glu ThrGlu Glu Glu Tyr Leu Asn Cys Phe Lys 1115 1120 1125 Ser Pro Ala Pro AlaSer Val Val Glu Tyr Leu Asn Thr Ser His 1130 1135 1140 Thr Gln Leu LeuSer Thr Lys Pro Phe Phe Ser Met Asp Asn Pro 1145 1150 1155 Asp Tyr GlnGln Asp Phe Cys Pro Leu Glu Leu Lys Thr His Thr 1160 1165 1170 Asn GlyHis Leu Pro Ala Ala Gln Asn Gln Glu Tyr Met Gly Leu 1175 1180 1185 GluVal His 1190

What is claimed is:
 1. A vaccine comprising a molecular homolog havingsufficient structural similarity to a tumor specific proteinendogenously expressed in a tumor such that the molecular homolog iscapable of inducing an immune response to the tumor specific protein ina subject bearing the tumor.
 2. The vaccine of claim 1 wherein themolecular homolog is a xenogeneic homolog of the tumor specific protein.3. The vaccine of claim 1 wherein the molecular homolog is generatedusing genetic engineering.
 4. The vaccine of claim 1 wherein themolecular homolog is a DNA molecule.
 5. The vaccine of claim 1 whereinthe molecular homolog is a-protein molecule.
 6. The vaccine of claim 1wherein the molecular homolog is attached to a virus as a carrier. 7.The vaccine of claim 6 wherein the virus is either the Adenovirus or theLentivirus.
 8. The vaccine of claim 1 wherein the tumor specific proteinis a tumor receptor.
 9. The vaccine of claim 8 wherein the tumorreceptor is an epidermal growth factor receptor (EGFR).
 10. The vaccineof claim 9 wherein the structural similarity between the molecularhomolog and EGFR ranges from 30-95%.
 11. The vaccine of claim 1 whereinthe molecular homolog is modified by attaching thereto a nanoparticle inorder to enhance the target specifity of the vaccine.
 12. The vaccine ofclaim 11 whererin the nanoparticle is an Adenovirus.
 13. The vaccine ofclaim 12 wherein the Adenovirus is modified by the peptide RGD.
 14. Thevaccine of claim 1 wherein the subject is an animal.
 15. The vaccine ofclaim 1 wherein the subject is a human.
 16. The vaccine of claim 9wherein the tumor is selected from the group consisting of mammarycancer, lung cancer, melanoma, hepatocarcinoma, fibrosarcoma, ovariancancer, colorectal cancer, prostate cancer, stomach cancer, bladdercancer, head and neck squamocarcinoma, and glioma.
 17. A vaccinesuitable for administering to a human or animal to inhibit growth orformation of a tumor comprsing a molecular homolog having sufficientstructural similarity to a tumor specific protein endogenously expressedin the tumor such that the molecular homolog is capable of inducing animmune response to the tumor specific protein in the subject.
 18. Thevaccine of claim 17, further comprising an pharmaceutically acceptablecarrier.
 19. The vaccine of claim 1, further comprising a nanoparticlewhich provides targeted modification of the vaccine.
 20. The vaccine ofclaim 19 wherein the nanoparticle has a diameter under 500 nm.
 21. Thevaccine of claim 20 wherein the nanoparticle has a diameter between 200nm-500 nm.
 22. The vaccine of claim 20 wherein the nanoparticle has adiameter between 100-200 nm.
 23. The vaccine of claim 20 wherein thenanoparticle has a diameter between 50-100 nm.
 24. The vaccine of claim19 wherein the nanoparticle is selected from group consisting ofliposome, PLGA, and Mannan-mofied Adenovirus.
 25. A cell capable ofexpressing the vaccine of claim
 1. 26. A commensal bacteria celltransformed stably with a DNA molecule coding a molecular homolog havingsufficient structural similarity to a tumor specific proteinendogenously expressed in a tumor such that the molecular homolog iscapable of inducing an immune response to the tumor specific protein ina subject bearing the tumor.
 27. A live vaccine comprising the commensalbacteria cell of claim 26
 28. A cellular vaccine comprising the cell ofclaim 25, the cellular vaccine is capable of inducing an immune responseagainst a tumor specific protein endogenously expressed in a tumor, whenthe cellular vaccine is administering to a subject bearing the tumor.29. A method of making a vaccine for inducing an immune response againsta tumor specific protein endogenously expressed in a tumor, comprisingselecting a molecular homolog having sufficient structural similarity tothe tumor specific protein so as to enable the molecular homolog toinduce the immune response against the tumor specific protein in thesubject bearing the tumor.
 30. The method of claim 29 wherein themolecular homolog is a xenogeneic homolog of the tumor specific protein.31. The method of claim 29 wherein the molecular homolog is generatedusing genetic engineering.
 32. The method of claim 29 wherein themolecular homolog is a DNA molecule.
 33. The method of claim 29 whereinthe molecular homolog is a protein molecule.
 34. The method of claim 29wherein the molecular homolog is attached to a virus as a carrier. 35.The method of claim 34 wherein the virus is either the Adenovirus or theLentivirus.
 36. The method of claim 29 wherein the tumor specificprotein is a tumor receptor.
 37. The method of claim 36 wherein thetumor receptor is an epidermal growth factor receptor (EGFR).
 38. Themethod of claim 37 wherein the structural similarity between themolecular homolog and EGFR ranges from 30-95%.
 39. The method of claim29 wherein the molecular homolog is modified by attaching thereto ananoparticle in order to enhance the target specifity of the vaccine.40. The method of claim 39 whererin the nanoparticle is an Adenovirus.41. The method of claim 40 wherein the Adenovirus is modified by thepeptide RGD.
 42. The method of claim 1 wherein the subject is an animal.43. The method of claim 1 wherein the subject is a human.
 44. A methodof inhibiting in vitro growth of tumor cells expressing a tumor specificprotein endogenously, comprising incubating with the tumor cells amolecular homolog having sufficient structural similarity to the tumorspecific protein such that the molecular homolog is capable of inbitingthe growth of the tumor cells, and measuring that growth of the tumorcells is inhibited.
 45. A method of inhibiting formation or growth of atumor of a subject, the tumor having a tumor specific proteinendogenously expressed therein, comprising the step of administering tothe subject a molecular homolog having sufficient structural similarityto the tumor specific protein so as to enable the molecular homolog toinduce an immune response to the tumor specific protein.
 46. The methodof claim 45 wherein the molecular homolog is a xenogeneic homolog of thetumor specific protein.
 47. The method of claim 45 wherein the molecularhomolog is generated using genetic engineering.
 48. The method of claim45 wherein the molecular homolog is a DNA molecule.
 49. The method ofclaim 45 wherein the molecular homolog is a protein molecule.
 50. Themethod of claim 45 wherein the molecular homolog is attached to a virusas a carrier.
 51. The method of claim 50 wherein the virus is either theAdenovirus or the Lentivirus.
 52. The method of claim 45 wherein thetumor specific protein is a tumor receptor.
 53. The method of claim 52wherein the tumor receptor is an epidermal growth factor receptor(EGFR).
 54. The method of claim 53 wherein the structural similaritybetween the molecular homolog and EGFR ranges from 30-95%.
 55. Themethod of claim 45 wherein the molecular homolog is modified byattaching thereto a nanoparticle in order to enhance the targetspecifity of the vaccine.
 56. The method of claim 55 whererin thenanoparticle is an Adenovirus.
 57. The method of claim 56 wherein theAdenovirus is modified by the peptide RGD.
 58. The method of claim 45wherein the subject is an animal.
 59. The method of claim 45 wherein thesubject is a human.
 60. A method of inducing regression of an existingtumor of a subject, the tumor having a tumor specific proteinendogenously expressed therein, comprising the step of administering tothe subject a molecular homolog having sufficient structural similarityto the tumor specific protein so as to enable the molecular homolog toinduce an immune response to the tumor specific protein
 61. A method ofinducing cytotoxic T-lymphocyte activity specifically directed against atumor cell expressing a tumor specific protein in a subject which isendogenously expressed in the tumor comprising administering to saidsubject a molecular homolog having sufficient structural similarity tothe tumor specific protein so as to enable the molecular homolog toinduce cytotoxic T-lymphocyte activity against the tumor specificprotein in the subject bearing the tumor.
 62. A method for inducingimmunity against a tumor specific protein endogenously expressed in atumor, comprising administering to a subject bearing the tumor amolecular homolog having sufficient structural similarity to the tumorspecific protein so as to enable the molecular homolog to induce theimmunity against the tumor specific protein.
 63. The method of claim 62wherein the molecular homolog is a xenogeneic homolog of the tumorspecific protein.
 64. The method of claim 62 wherein the molecularhomolog is generated using genetic engineering.
 65. The method of claim62 wherein the molecular homolog is a DNA molecule.
 66. The method ofclaim 62 wherein the molecular homolog is a protein molecule.
 67. Themethod of claim 62 wherein the molecular homolog is attached to a virusas a carrier.
 68. The method of claim 67 wherein the virus is either theAdenovirus or the Lentivirus.
 69. The method of claim 62 wherein thetumor specific protein is a tumor receptor.
 70. The method of claim 69wherein the tumor receptor is an epidermal growth factor receptor(EGFR).
 71. The method of claim 70 wherein the structural similaritybetween the molecular homolog and EGFR ranges from 30-95%.
 72. Themethod of claim 62 wherein the molecular homolog is modified byattaching thereto a nanoparticle in order to enhance the targetspecifity of the vaccine.
 73. The method of claim 72 whererin thenanoparticle is an Adenovirus.
 74. The method of claim 73 wherein theAdenovirus is modified by the peptide RGD.
 75. The method of claim 62wherein the subject is an animal.
 76. The method of claim 62 wherein thesubject is a human.
 77. A method of immunizing an animal against a tumorhaving a tumor specific protein endogenously expressed therein,comprising the step of administering to the animal a molecular homologhaving sufficient structural similarity to the tumor specific protein soas to enable the molecular homolog to induce an immune response to thetumor specific protein.
 78. The method of claim 77 wherein the animal isa mammal.
 79. The method of claim 77 wherein the animal is an avianorganism.
 80. The method of claim 79 wherein the avian organism is achicken.
 81. The method of claim 77 wherein the animal is a mouse. 82.The method of claim 78 wherein the mammal is a human.
 83. The method ofclaim 77 wherein the administering is subcutaneous.
 84. The method ofclaim 77 wherein the administering is intradermal.
 85. The method ofclaim 77 wherein the administering is intravenous.
 86. The method ofclaim 77 wherein the administering is intraperitoneal.
 87. The vaccineof claim 77 wherein the molecular homolog is a xenogeneic homolog of thetumor specific protein.
 88. The vaccine of claim 77 wherein themolecular homolog is generated using genetic engineering.
 89. Thevaccine of claim 77 wherein the molecular homolog is a DNA molecule. 90.The vaccine of claim 77 wherein the molecular homolog is a proteinmolecule.
 91. The vaccine of claim 77 wherein the molecular homolog isattached to a virus as a carrier.
 92. The vaccine of claim 91 whereinthe virus is either the Adenovirus or the Lentivirus.
 93. The vaccine ofclaim 77 wherein the tumor specific protein is a tumor receptor.
 94. Thevaccine of claim 93 wherein the tumor receptor is an epidermal growthfactor receptor (EGFR).
 95. The vaccine of claim 94 wherein thestructural similarity between the molecular homolog and EGFR ranges from30-95%.
 96. The vaccine of claim 77 wherein the molecular homolog ismodified by attaching thereto a nanoparticle in order to enhance thetarget specifity of the vaccine.
 97. The vaccine of claim 96 whererinthe nanoparticle is an Adenovirus.
 98. The vaccine of claim 97 whereinthe Adenovirus is modified by the peptide RGD.